http://www.bbc.co.uk/health/talking/tests/blood_full_blood_count.shtml.Parasites on blood film and marrow
Assessment of vitamin B12 and folate status
Erythrocyte sedimentation rate
Investigation of haemolytic anaemia
Urobilin, urobilinogen, and urinary haemosiderin
Hereditary haemolytic anaemias
Investigation of possible thalassaemia
Investigation of sickle haemoglobin
Estimation of haemoglobin A2 (α2δ2)
Estimation of fetal haemoglobin
Testing for unstable haemoglobins
Molecular tests for diagnosis of thalassaemia
Immunophenotyping for GPI-linked proteins
Activated partial thromboplastin time
►► Disseminated intravascular coagulation
Antithrombin and proteins C and S
Cytochemistry tests (leukaemia diagnosis)
Neutrophil alkaline phosphatase
Bacterial contamination of blood products
Cytogenetics: prenatal diagnosis
Cytogenetics: haematological malignancies
Human leucocyte antigen (tissue) typing
Polymerase chain reaction amplification of DNA
In situ hybridization and fluorescence in situ hybridization
Called complete blood count (CBC) in the United States (USA).
Before the advent of modern haematology blood analysers, the blood count consisted of a haemoglobin (Hb) concentration (estimated using a manual colorimetric technique), a white cell count, and a manual platelet count. Other parameters, such as mean cell volume (MCV), had to be mathematically calculated (derived) using the measured variables Hb, red cell count (RCC), and packed cell volume (PCV).
Modern analysers use a variety of methods to provide a huge range of full blood count (FBC) variables, including electronic impedance, laser light scatter, light absorbance, and staining characteristics. The resultant FBC provides measured variables such as Hb, PCV, and RCC, along with derived (mathematically) MCV, mean cell Hb (MCH), and mean corpuscular Hb concentration (MCHC). These machines also provide automated platelet counts and a 5-part differential white blood count (WBC).
Sample: peripheral blood ethylenediamine tetra-acetic acid (EDTA); the sample should be analysed in the laboratory within 4h, if possible.
•Red cell distribution width (RDW).
Some machines are even more sophisticated and will measure reticulocyte counts, in addition to determination of reticulocyte Hb and MCV.
Why ask for an FBC? How will this aid the diagnosis or management of the patient? The FBC assesses several different parameters and can provide a great deal of information. The red cell variables will determine whether or not the patient is anaemic. If anaemia is present, the MCV is likely to provide clues as to the cause of the anaemia. The white cells are often raised in infection—neutrophilia in bacterial infections and lymphocytosis in viral (but not always so). Platelets (size or number) may be abnormal either as a direct effect of an underlying blood disease or simply reflecting the presence of some other underlying pathology. Most of us take a somewhat cursory glance at the FBC when the report arrives on the ward or in clinic, but a more detailed look may reveal a great deal more!
http://www.bbc.co.uk/health/talking/tests/blood_full_blood_count.shtml.
http://www.rcpa.edu.au/pathman/full_blo.htm.
Units: g/dL or g/L (Europe uses SI units; the USA uses g/dL or grams %).
Defines anaemia (Hb < lower limit of normal, adjusted for age and sex). Values differ between ♂ and ♀, since androgens drive RBC production and hence an adult ♂ has higher Hb, PCV, and RCC than an adult ♀.
Useful in the diagnosis of polycythaemic disorders (↑ production of microcytic, hypochromic erythrocytes) and thalassaemias.
•Hypoproliferative anaemias, e.g. iron, vitamin B12, and folate deficiencies.
•Aplasias, e.g. idiopathic or drug-induced (do not forget chemotherapy).
•Parvovirus B19 infection-induced red cell aplasia resulting in transient marked anaemia.
Unit: femtolitre (fL), 10−15L.
Provided as part of the derived variables or, if you know the PCV and RCC, can be calculated as (PCV/RCC), e.g. if the PCV is 0.45 and RCC 5 × 1012/L, then the MCV is 90fL.
This index provides a useful starting point for the evaluation of anaemia (see Table 3.1).
| MCV ↓ | MCV normal | MCV ↑ |
| Iron deficiency | Blood loss | B12 or folate deficiency |
| Thalassaemia homo-/heterozygotes | Myelodysplasia | Myelodysplasia |
| Sideroblastic anaemia | Anaemia of chronic disease |
Unit: pg.
•High (for range, 
Normal ranges, inside front cover): macrocytosis.
•Low: microcytosis, e.g. iron deficiency anaemia.
Unit: g/dL or g/L.
•Of value in evaluation of microcytic anaemias.
•High: severe prolonged dehydration, hereditary spherocytosis, cold agglutinin disease.
•Low: iron deficiency anaemia, thalassaemia.
These are not entirely synonymous terms (but they are more or less). If blood is placed in a microcapillary tube and centrifuged, the red cells are spun down to the bottom, leaving the plasma above. The RBCs will occupy about 40% of the blood in the tube—the blood will have a PCV of 0.4 (or 40%). The Hct is similar, but derived, using automated blood counters.
PCV unit: L/L (although the units are seldom cited in reports).
•High PCV: polycythaemia (any cause).
•Low PCV: anaemia (any cause).
Measures the range of red cell size in a sample of blood, providing information about the degree of red cell anisocytosis, i.e. how much variation there is between the sizes of the red cells. Of value in some anaemias, e.g.:
•↓ MCV with normal RDW suggests β-thalassaemia trait.
•↓ MCV with high RDW suggests iron deficiency.
(Probably noticed more by haematology staff than those in general medicine!)
Defined as red cells with Hb <280g/L. This is useful in identifying patients with functional iron deficiency and monitoring response to erythropoietin-stimulating agent (ESA) Epo therapy and the requirement for IV iron.
Predict iron deficiency anaemia and functional iron deficiency, and predict response to IV iron in those on haemodialysis.
Assessment of iron status, pp. 244–247.
The automated differential white cell count is provided as part of the FBC. The RBCs in the sample are lysed before WBCs are counted. A typical FBC will show the total WBC and the 5-part differential white cell count, broken down into the five main white cell subtypes in peripheral blood which include:
The printed FBC usually shows the % of each type of white cell, but unless the absolute WBC (as × 109/L) is known, this % count is of little value.
► As a general rule, ignore the % count—you cannot detect abnormalities, such as neutropenia, unless you have the absolute values.
Abnormalities of the WBC, e.g. neutrophilia, neutropenia, etc., are discussed in OHCM 10e, p. 330.
Platelets (thrombocytes in the USA) are the smallest cells in peripheral blood. Traditional counting methods with a microscope and counting chamber have now been replaced by automated counting with haematology analysers.
This is analogous to the RDW and provides information about the range of platelet sizes in a blood sample.
•The platelet distribution width (PDW) will be high if there are giant platelets in the presence of normal-sized platelets, e.g. essential thrombocythaemia (one of the myeloproliferative disorders).
•The PDW will be normal in a reactive thrombocytosis (where the platelet count is ↑, but they are all of normal size).
This is an in vitro artefact in some individuals. Platelets clump in EDTA and the blood analyser will report spurious thrombocytopenia. The actual in vivo count is normal and the platelets function normally. Taking blood into citrate or heparin will show the patient’s platelet count to be normal. The presence of even a small blood clot in an EDTA sample may also reduce the platelet count (the haematology technical staff will usually check to see whether the sample contains a small clot before sending out the report).
Examining a stained peripheral blood smear under the microscope allows the examination of red cells, white cells, and platelets (see Fig. 3.1). In addition, the blood film will help detect parasites (e.g. malaria, trypanosomes) or abnormal cells in the blood.
The haematology laboratory will usually examine a peripheral blood film if the patient’s indices are abnormal (unless there has been no major change from previous FBCs). If you suspect an underlying blood disorder, you should request a film. Note: the laboratory staff may not make a film if the indices are completely normal.
A fingerprick blood sample may be spread onto a glass slide (the phlebotomist may do this for you), air-dried, fixed, and stained. Alternatively, a drop of EDTA blood may be treated in the same manner (the haematology laboratory staff will make the film). Beware: old EDTA samples produce strange artefacts such as extreme red cell crenation—if a film is required, it should be made from a fresh blood sample.
•Sample: EDTA (as fresh as possible).
•Membrane changes (e.g. oxidative membrane damage).
•Inclusions, e.g. Howell–Jolly bodies, malarial parasites, haemoglobin C (HbC) crystals, etc.
•Abnormalities such as toxic granulation, dysplastic changes.
•Presence of abnormal cells, e.g. leukaemic blasts or lymphoma cells.
•Red cell rouleaux (stacking effect—seen, e.g. when ESR is ↑).
•Occasionally see circulating carcinoma cells.
OHCM 10e, p. 328.
Fig. 3.1 Normal peripheral blood film showing a neutrophil with its typical lobulated nucleus, numerous red cells, and a few platelets.
In health, the normal RBC is a pink, biconcave disc-shaped cell, and most red cells are roughly the same size, shape, and colour. They should be roughly the size of a small lymphocyte nucleus. Many diseases and deficiency disorders alter the RBC appearance by either reducing its Hb content or altering the membrane such that characteristic morphological abnormalities are produced. Examples include target cells, sickle cells, bite cells, burr cells, and many others (see Table 3.2). Most of the morphological features are not absolutely specific for one particular disorder, but rather they suggest a range of conditions that may be associated with the RBC feature (see Fig. 3.2). This should prompt you to look for conditions which might account for the abnormality.
► Pay attention to the peripheral blood film comment (inserted on the report by the haematology laboratory staff or automated blood counter)—it should help you decide which tests to carry out next. Conversely, cryptic laboratory comments like ‘anisopoikilocytosis noted’ do not help the clinician much. (Note: aniso = unequal; poikilo = varied.)
Table 3.2 Peripheral blood film in anaemias
| Microcytic RBCs | Fe deficiency, thalassaemia trait and syndromes, congenital sideroblastic anaemia, ACD (if long-standing) |
| Macrocytic RBCs | Alcohol/liver disease (round macrocytes), MDS, pregnancy and newborn, compensated haemolysis, B12 or folate deficiency, hydroxyurea and antimetabolites (oval macrocytes), acquired sideroblastic anaemia, hypothyroidism, chronic respiratory failure, aplastic anaemia |
| Dimorphic RBCs | Two populations, e.g. Fe deficiency responding to Fe, mixed Fe and B12/folate deficiencies, sideroblastic anaemia, post-red cell transfusion |
| Hypochromic RBCs | Reduced Hb synthesis, e.g. Fe deficiency, thalassaemia, sideroblastic anaemia |
| Polychromatic RBCs | Blood loss or haematinic treatment, haemolysis, marrow infiltration |
| Spherocytes | Hereditary spherocytosis, haemolysis, e.g. warm AIHA, delayed transfusion reaction, ABO, HDN, DIC, and MAHA, post-splenectomy |
| Pencil/rod cells | Fe deficiency anaemia, thalassaemia trait and syndromes, PK deficiency |
| Elliptocytes | Hereditary elliptocytosis, MPD, and MDS |
| Fragmented RBCs | MAHA, DIC, renal failure, HUS, TTP |
| Teardrop RBCs | Myelofibrosis, metastatic marrow infiltration, MDS |
| Sickle cells | Sickle-cell anaemia, other sickle syndromes (not sickle trait) |
| Target cells | Liver disease, Fe deficiency, thalassaemia, HbC syndromes |
| Crenated RBCs | Usually storage or EDTA artefact. Genuine RBC crenation may be seen post-splenectomy and in renal failure (→ burr cells) |
| Burr cells | Renal failure |
| Acanthocytes | Hereditary acanthocytosis, a-β-lipoproteinaemia, McLeod red cell phenotype, PK deficiency, chronic liver disease (especially Zieve’s syndrome) |
| Bite cells | G6PD deficiency, oxidative haemolysis |
| Basophilic stippling | Megaloblastic anaemia, lead poisoning, MDS, liver disease, haemoglobinopathies, e.g. thalassaemia |
| Rouleaux | Chronic inflammation, paraproteinaemia, myeloma |
| ↑ reticulocytes | Bleeding, haemolysis, marrow infiltration, severe hypoxia, response to haematinic therapy |
| Heinz bodies | Not seen in normals (removed by spleen), small numbers seen post-splenectomy, oxidant drugs, G6PD deficiency, sulfonamides, unstable Hb (Hb Zurich, Köln) |
| Howell–Jolly bodies | Composed of DNA, removed by the spleen, seen in dyserythropoietic states, e.g. B12 deficiency, MDS, post-splenectomy, hyposplenism |
| H bodies | HbH inclusions, denatured HbH (β4 tetramer), stain with methylthioninium chloride (methylene blue), seen in HbH disease (– –-/–α), less prominent in α-thalassaemia trait, not present in normal subjects |
| Hyposplenic film | Howell–Jolly bodies, target cells, occasional nucleated RBCs, lymphocytosis, macrocytosis, acanthocytes. Infectious mononucleosis, any viral infection, toxoplasmosis, drug reactions |
ABO, ABO blood groups; ACD, anaemia of chronic disease; AIHA, autoimmune haemolytic anaemia; DIC, disseminated intravascular coagulation; Fe, iron; G6PD, glucose-6-phosphate dehydrogenase; HDN, haemolytic disease of the newborn; HUS, haemolytic uraemic syndrome; MAHA, microangiopathic haemolytic anaemia; MPD, myeloproliferative disease; PK, pyruvate kinase; TTP, thrombotic thrombocytopenic purpura.
Although there are now highly sensitive monoclonal antibody (MoAb) kits for the diagnosis of diseases such as malaria, a well-stained blood film can often make the diagnosis more easily and more cheaply. Blood films are useful for confirming a diagnosis of:
Some diseases, such as leishmaniasis, require bone marrow aspiration and staining (in fact, there are many infections that can be diagnosed using bone marrow):
•Tropheryma whippelii (Whipple’s disease).
See Fig. 3.3 for examples.
Fig. 3.3 Parasites, such as malaria, loa loa, and trypanosomes, may be seen on a stained blood film.
MedlinePlus (2016). CBC blood test. http://www.nlm.nih.gov/medlineplus/ency/article/003642.htm.
Thomas DW, Hinchliffe RF, Briggs C, Macdougall IC, Littlewood T, Cavill I; British Committee for Standards in Haematology. Guideline for the laboratory diagnosis of functional iron deficiency. Br J Haematol 2013; 161: 639–48.
In much the same way as RBC morphology provides clues about underlying disease, so too does microscopical examination of stained peripheral blood WBCs. Modern counters enumerate WBCs, and our greater reliance on modern technology means that visual inspection of blood films is becoming a dying art. A well-stained blood film may provide the diagnosis much more cheaply (see Fig. 3.4).
•Sometimes difficult to determine the diagnosis since so few WBCs.
•May suggest B12 or folate deficiency (are the RBCs normal or large?).
•Aplastic anaemia: are the platelets and Hb normal?
•Underlying leukaemia: are there any leukaemic blasts* present?
•Overwhelming infection: may see toxic granulation (large, dark granules in the cytoplasm—not diagnostic, but suggestive).
•May be immune or post-viral: atypical lymphocytes may be seen; other indices usually normal.
Note: a blast (*) is a primitive cell seen in the marrow in large numbers in leukaemia. We all have some blasts in our marrows, but these should be <5% of the total nucleated bone marrow cells in health.
•Lymphocytes: suggests viral, CLL, acute leukaemia (lymphoblastic).
•Granulocytic? (neutrophils, eosinophils, basophils)—may be reactive or CML.
•Abnormal-looking WBC? Look for Auer rods (≡ AML), smear cells (CLL), bilobed neutrophils (pseudo-Pelger cells seen in MDS).
(See Table 3.3.)
•Viral illnesses often produce bizarre films in children, but beware of complacency (acute leukaemia may be the cause).
•MDS and malignancies like CLL and CML are diseases of older individuals.
•May be worth repeating the FBC and film to see if abnormalities have resolved.
•If the patient is unwell or has lymphadenopathy or hepatosplenomegaly, then an underlying disease must be excluded.
Table 3.3 Some WBC abnormalities seen on FBC reports
| Atypical lymphocytes | Infectious mononucleosis, any viral infection, toxoplasmosis, drug reactions |
| Auer rods | Seen in myeloblasts; pathognomonic of AML Prominent in AML M3 subtype (acute promyelocytic leukaemia) |
| Pelger–Huët anomaly | Bilobed neutrophils. May be hereditary (neutrophils are functionally normal) or acquired, e.g. MDS (pseudo-Pelger cells) |
| Left-shifted | Immature WBCs seen in peripheral blood. Seen in severe infections, inflammatory disorders, DKA, marrow ‘stress’, MPD, CML |
| Right-shifted | Hypermature WBCs seen in, e.g. megaloblastic anaemia and iron deficiency |
| Toxic granulation | Coarse granules in neutrophils. Seen in severe infection, post-operatively, and inflammatory disorders |
| Smear cells | Lymphocytes in which the cell membrane has ruptured when making the blood film—there are no smear cells in vivo! Seen in CLL |
Fig. 3.4 Blood film: atypical WBCs (this was from a patient with glandular fever, but these cells may be seen in any viral illness).
Anaemia of iron deficiency is caused by defective synthesis of Hb, resulting in red cells that are smaller than normal (microcytic) and contain reduced amounts of Hb (hypochromic). The diagnosis of iron deficiency anaemia is generally straightforward, but it may be confused with anaemia of chronic disease (ACD) or other hypochromic anaemias (see Table 3.4 and Fig. 3.5).
Iron plays a pivotal role in many metabolic processes, and the average adult contains between 3 and 5g of iron, of which two-thirds are present in the O2-carrying molecule Hb. Somewhat surprisingly, there is no specific excretion mechanism in humans. Iron balance is controlled at the level of gut absorption and relies on two iron-sequestering proteins transferrin (iron transport and recycling of iron) and ferritin (safeguards iron entry into the body and maintains surplus iron in a safe and readily accessible form).
This is the 1° iron storage protein, consisting of 24 apoferritin subunits forming a hollow sphere (each can hold up to 4500 iron atoms).
Haemosiderin, located predominantly in macrophages, is a water-soluble protein–iron complex with an amorphous structure.
Transferrin contains only 4mg of iron and is the principal iron transport protein with >30mg of iron transported round the body daily. Synthesis of transferrin is inversely proportional to the body iron stores, with ↑ transferrin concentration when iron stores are reduced.
The transferrin receptor (TfR) is a disulfide-linked dimer, composed of two identical 85kDa subunits. The serum TfR (sTfR) concentration is elevated in iron deficiency. However, sTfR may also ↑ in any condition in which there is ↑ erythropoiesis, e.g. haemolytic anaemias, thalassaemia, polycythaemia vera, and other myeloproliferative disorders.
•Serum iron and transferrin (as TIBC).
•% hypochromic cells in peripheral blood.
•Reticulocyte MCH (CHr) and reticulocyte Hb content (Ret-He).
•Red cell protoporphyrin assay (not widely available).
•Bone marrow aspirate (stained for iron)—the ‘gold standard’.
Remember, iron deficiency is not an ‘all-or-nothing’ phenomenon. In progressive deficiency, there is a gradual loss of iron with subtle alterations of iron-related parameters, during which the red cells may look entirely normal. In the initial stages of developing iron deficiency, macrophages become depleted of iron and the serum ferritin ↓ to the lower end of the normal range; during this ‘latency’ period, the Hb is normal. As the deficiency progresses, plasma iron levels ↓ and TIBC ↑. Free RBC protoporphyrin levels ↑ as it accumulates, and eventually hypochromic RBCs appear in the peripheral blood. At this stage, an FBC will usually show ↓ Hb, MCV, MCH, and MCHC, and the peripheral blood film will show microcytic hypochromic red cells.
(See Fig. 3.6.)
•Serum iron ↓ and TIBC ↑ (generally unhelpful and little used today).
•Microcytic and hypochromic RBCs on blood film.
►Beware: serum ferritin is an acute phase protein and may be normal or even ↑ in inflammatory, malignant, or liver disease. During the inflammatory response, the iron/TIBC are unlikely to be of any value (iron ↓ and TIBC will be ↓). If an inflammatory process is suspected, an alternative test is required, e.g. STfR, which is not affected by inflammatory disorders.
Fig. 3.6 The % hypochromic red cells (provided by some automated counters) helps in the diagnosis of iron deficiency. Note that the RBC volume and Hb content (HC) are both shifted to the LEFT (= small, pale red cells).
Table 3.4 Hypochromic anaemias—may be confused with iron deficiency
| Disorder | Example |
| Disorders of iron metabolism | Iron deficiency anaemia: |
| Anaemia of chronic disorders | |
| Disorders of haem synthesis | Sideroblastic anaemias: |
| Globin synthesis disorders | Thalassaemias: |
This is the situation where iron is retained in body stores and, although stores are adequate, delivery to the bone marrow is inadequate for erythropoiesis. It occurs in inflammatory disease, being one component of ACD. Functional iron deficiency also occurs during the use of Epo where response is improved with the use of IV iron.
Can be iatrogenic or genetic (haemochromatosis).
Ferritin and transferrin saturation are elevated. Investigations to assess the degree of cardiac and hepatic iron accumulation are required.
OHCM 10e, p. 326.
Iron Disorders Institute. Iron deficiency anemia. http://www.irondisorders.org/iron-deficiency-anemia.
Iron Panel. http://www.ironpanel.org.au/AIS/AISdocs/adultdocs/Acontents.html.
Provan D, Weatherall D. Acquired anaemias and polycythaemia. Lancet 2000; 355: 1260–8.
Measurement of serum B12 and red cell folate levels is necessary in the investigation of macrocytic anaemia and certain other situations (see below). Serum folate levels are an unreliable measurement of body stores of folate—the red cell folate level is probably more meaningful.
•Serum and red cell folate units: µg/L.
•Sample: clotted blood sample (serum B12 and folate) and peripheral blood EDTA (red cell folate).
Deficiency of either vitamin leads to megaloblastic anaemia where there is disruption of cell division in all actively dividing cells (includes the bone marrow and gut). In the marrow, there is nuclear:cytoplasmic asynchrony where the nuclei are immature despite a mature, well-haemoglobinized cytoplasm. In the peripheral blood, there may be anaemia, often with pancytopenia; the red cells show oval macrocytic changes with basophilic stippling and occasionally nucleated red cells. Neutrophils typically become hypersegmented (they have >5 lobes).
Until recently, B12 and folate assays were tedious microbiological assays, but these have now been replaced by automated techniques using radioisotopic methods, which allow large numbers of samples to be batched and tested fairly cheaply.
In the past, deficiency of either B12 or folate was synonymous with macrocytic anaemia, but deficiency of either vitamin may present without anaemia or macrocytosis—remember, these are late features of the disease. However, in most cases of deficiency, the marrow will show characteristic megaloblastic change (nuclear asynchrony with giant metamyelocytes). ► Deficiency of B12 may cause neurological problems in the absence of anaemia.
•Patients with gastrointestinal tract (GIT) disease, glossitis, abnormalities of taste, previous surgery, or radiotherapy to the stomach or small bowel.
•Neurological disease, e.g. peripheral neuropathy, demyelination.
•Psychiatric disturbance, e.g. confusion, dementia.
•Malnutrition, e.g. growth impairment in children; vegans.
•Autoimmune disease of the thyroid, parathyroid, or adrenals.
•Patients with a family history of pernicious anaemia.
•Others, e.g. drugs that interfere with vitamin absorption or metabolism such as nitrous oxide, phenytoin, etc.
► B12 and folate deficiencies produce similar clinical and laboratory features:
•Hypersegmented neutrophils (also seen in renal failure, iron deficiency, and MDS).
(See Fig. 3.7.)
Fig. 3.7 Blood film of megaloblastic anaemia. There are large oval macrocytes and two hypersegmented neutrophils (the nucleus has >5 lobes).
•Serum and red cell folate levels.
•Intrinsic factor antibodies (IFA), +ve in 50–75% of patients with PA.
•Consider the bone marrow (helps exclude MDS, myeloma, and other pathologies that give rise to macrocytic anaemia, but seldom performed today since it is easy to get B12 and folate results back quickly).
Normal ranges are based on two standard deviations either side of the mean, so there will be ‘normal’ people who have ‘abnormal’ B12 (or folate) levels.
The lowest levels are seen in those most deficient. What matters is whether there is tissue deficiency (leads to marrow and neurological changes).
Difficult, but common! Probably worth repeating the test and reviewing the patient and other results. If no evidence of tissue deficiency, can probably observe the patient. If there is evidence of tissue deficiency, then the patient will require treatment.
The most reliable method is probably the measurement of serum homocysteine (accumulates in vitamin B12 and folate deficiency).
•Transcobalamin I deficiency (very rare).
↓ level seen in hospitalized patients due to −ve folate balance.
•Parietal cell (+ve in serum of 90% of patients with PA, but also found in other disorders and 15% of the normal elderly) and intrinsic factor antibodies (IFA now preferred—if +ve, confirms diagnosis of PA).
•Schilling test (urinary excretion method where the addition of intrinsic factor (IF) restores B12 absorption in PA, but not in intestinal, e.g. ileal, disease), seldom performed now due to lack of required radioisotope, or
•Endoscopy with duodenal biopsy.
•Other gastroenterology tests for malabsorption ( Gastroenterology, p. 522).
•Endoscopy with duodenal biopsy.
•Other gastroenterology tests for malabsorption ( Gastroenterology, p. 522).
OHCM 10e, p. 266.
Devalia V, Hamilton MS, Molloy AM; British Committee for Standards in Haematology. Guidelines for the diagnosis and treatment of cobalamin and folate disorders. Br J Haematol 2014; 166: 496–513.
Guidelines and Protocols Advisory Committee (2012). Cobalamin (vitamin B12) deficiency: investigation and management. http://www2.gov.bc.ca/gov/content/health/practitioner-professional-resources/bc-guidelines/vitamin-b12.
Hoffbrand AV. Megaloblastic anaemia and miscellaneous deficiency anaemias. In: DA Warrell, TM Cox, JD Firth, eds. Oxford Textbook of Medicine, 5th edn. Oxford: Oxford University Press, 2010; pp. 4402–19.
Provan D, Weatherall D. Acquired anaemias and polycythaemia. Lancet 2000; 355: 1260–8.
This simple, but very useful, qualitative test measures how fast a patient’s red cells fall through a column of blood. It is a sensitive, but non-specific, index of plasma protein changes that result from inflammation or tissue damage. The ESR is affected by Hct variations, red cell abnormalities (e.g. poikilocytosis, sickle cells), and delay in analysis, and it is therefore less reliable than measurement of plasma viscosity (PV). The ESR is affected by age, sex, menstrual cycle, pregnancy, and drugs (e.g. OCP, steroids) (see Table 3.5).
The ESR is widely used in clinical medicine and, despite attempts (by haematology departments) to replace the ESR with PV, the ESR has remained in use and appears to retain a valuable place in the armoury of disease diagnosis and monitoring.
•Sample: peripheral blood EDTA; the sample should be analysed in the laboratory within 4h.
Many factors influence the ESR, causing a high or low result:
•High ESR (significant*—look for a cause):
•Any inflammatory disorder, e.g. infection, rheumatoid, TB.
•MI (the ESR ↑ as an early response).
Note: (*) depends on exactly how high. An ESR of 30 probably means little, but >100 is highly significant and indicates something seriously wrong.
•Low ESR (rarely important, but useful for exams):
►► A normal ESR does not exclude organic disease.
OHCM 10e, p. 372.
Brigden M. Clinical utility of the erythrocyte sedimentation rate. Am Fam Physician 1999; 60: 1443–50. http://www.aafp.org/afp/991001ap/1443.html.
Harris GJ. Plasma viscometry and ESR in the elderly. Med Lab Technol 1972; 29: 405–10.
Lewis SM. Erythrocyte sedimentation rate and plasma viscosity. Ass Clin Pathol Broadsheet 1982; 94: 1–6.
This test is a sensitive, but non-specific, index of plasma protein changes, which result from inflammation or tissue damage. Provides much the same information as the ESR. The ESR and PV tend to rise in parallel, but the PV is unaffected by Hct variations (e.g. severe anaemia or polycythaemia) and delay in analysis up to 24h, and it is therefore more reliable than the ESR. It is not affected by sex but is affected by age, exercise, and pregnancy. It is constant in health and shows no diurnal variation. There is a suggestion that the PV may be a more sensitive indicator of disease severity than the ESR.
•Sample: peripheral blood EDTA. The sample is centrifuged and the plasma removed.
•Normal range: 1.50–1.72cP (or mPA/s at 25°C).
A high PV generally signifies some underlying pathology, e.g. inflammatory states, paraproteinaemias such as MGUS or myeloma; low PV can be ignored.
Note: despite the advantages outlined, the PV has not been adopted by all medical staff (who still prefer the ESR as a measure of inflammation). The PV is better for monitoring hyperviscosity syndromes, e.g. Waldenström’s macroglobulinaemia. The fact that both tests are still used shows that there is a role for both.
OHCM 10e, p. 373.
Cooke BM, Stuart J. Automated measurement of plasma viscosity by capillary viscometer. J Clin Pathol 1988; 41: 1213–16.
This infection is caused by EBV. Infected cells produce so-called heterophile antibodies (these are IgM molecules that agglutinate horse and sheep RBCs but do not agglutinate ox RBCs and do not react at all with guinea pig RBCs).
There are various kits available that can detect the presence of heterophile antibodies and, in the right clinical context, will confirm a diagnosis of EBV infection. The Monospot test is probably the commonest in current use. The Paul–Bunnell test was the first to demonstrate the presence of heterophile antibodies in patients with EBV infection.
Glandular fever often affects young adults (12–25 years) and results in malaise, fever, tonsillitis, petechial haemorrhages on the palate, and lymphadenopathy. Splenomegaly is fairly common. A similar clinical picture is seen in CMV, Toxoplasma, and early HIV infections.
Hoff G, Bauer S. A new rapid slide test for infectious mononucleosis. JAMA 1996; 194: 351–3.
The normal red cell has a lifespan of ~120 days. Anaemia resulting from ↓ RBC lifespan is termed haemolytic. May be inherited or acquired, and the basic underlying mechanisms may involve abnormalities of the RBC membrane, RBC enzymes, or Hb.
Extravascular haemolysis implies RBC breakdown by the reticuloendothelial system (RES) (e.g. liver, spleen, and macrophages at other sites), whilst intravascular haemolysis describes RBC breakdown in the circulation itself (see Fig. 3.8). There are many investigations available that will help determine the predominant site of destruction, which, in turn, will help define the underlying cause of haemolysis, which is why we do the tests in the first place.
The main question is whether the patient’s anaemia is due to haemolysis or some other underlying mechanism such as blood loss, marrow infiltration, etc.
OHCM 10e, p. 336.
Fig. 3.8 Increased red cell breakdown may be extravascular (outside the circulation, predominantly the spleen, liver, and marrow) or intravascular (within the vessels).
Modified from Lewis SM, Bain BJ & Bates I, eds. (2001) Dacie & Lewis Practical Haematology, 11th edition, Churchill Livingstone, Edinburgh.
•Evidence of ↑ red cell destruction.
•Evidence of ↑ red cell production (to compensate for red cell loss).
•Evidence of autoantibody in the patient’s serum.
•↑ serum bilirubin (split conjugated/unconjugated is useful).
•↑ serum LDH (reflecting ↑ RBC turnover).
•Spherocytes or other abnormal RBCs, e.g. fragments on blood film.
•Plasma haptoglobins may be ↓ or absent.
•↑ faecal and urinary urobilinogen (faecal not measured).
•↓ RBC lifespan (seldom measured nowadays).
•↑ reticulocytes (on film, manual, or automated count). Not absolutely specific, will ↑ in brisk acute bleed, e.g. GIT.
•↑ MCV (reticulocytes are larger than mature RBCs, and do not forget folate deficiency, which occurs in haemolytic disorders).
•RBC morphology (e.g. spherocytes, elliptocytes).
•Non-immune: check RBC morphology (e.g. TTP/HUS).
•Is there some other underlying disease?
Hill QA, Stamps R, Massey E, Grainger JD, Provan D, Hill A; British Society for Haematology Guidelines. Guidelines on the management of drug-induced immune and secondary autoimmune, haemolytic anaemia. Br J Haematol 2017; 177: 208–20.
Hill QA, Stamps R, Massey E, Grainger JD, Provan D, Hill A; British Society for Haematology. The diagnosis and management of primary autoimmune haemolytic anaemia. Br J Haematol 2017; 176: 395–411.
Schick P (2016). Hemolytic anemia. http://www.emedicine.com/med/topic979.htm.
These are immature RBCs formed in the marrow and found in small numbers in normal peripheral blood. They represent an intermediate maturation stage in the marrow between the nucleated RBC and the mature RBC (the reticulocyte lacks a nucleus but retains some nucleic acid). Measuring the number of reticulocytes in the blood may help determine whether the anaemia is due to ↓ RBC production. The reticulocyte count is also a useful measure of response to haematinic (iron, B12, or folate) replacement therapy.
•Modern automated blood counters using laser technology measure the number of reticulocytes directly.
•Demonstrated by staining with supravital dye for the nucleic acid.
•Appear on blood film as larger than mature RBCs with fine, lacy blue staining strands or dots (see Fig. 3.9).
•Usually expressed as a percentage of total red cells, e.g. 5%, though absolute numbers can be derived from this and the total red cell count.
•Normal range: 0.5–2.5% (50–100 × 109/L).
Fig. 3.9 Blood film of numerous spherocytes (small, darker red cells) and reticulocytes (larger red cells) in autoimmune haemolytic anaemia.
•Response to oral iron therapy.
•Myeloproliferative disorders.
•Marrow recovery following chemotherapy or radiotherapy.
•Iron, folate, or B12 deficiency. Note: the return of reticulocytes is the earliest sign of response to replacement therapy.
•Immediately post-chemotherapy or radiotherapy.
•Autoimmune disease, especially refractory anaemia (RA).
Howells MR, Jones SE, Napier JA, Saunders K, Cavill I. Erythropoiesis in pregnancy. Br J Haematol 1986; 64: 595–9.
Haptoglobins (Hps) are plasma proteins synthesized by the liver, whose function is the removal of free plasma Hb. Hp molecules bind free Hb and are taken up by the RES for degradation. Hp–Hb complexes do not appear in the urine because their large size prevents them from passing through the renal tubules.
The Hp–Hb complex is cleared by the RES at a rate of 15mg/100mL/h, which means that even very mild haemolysis will cause the disappearance of Hps from the circulation. Serum Hps should be measured in patients with suspected intravascular haemolysis. However, the Hp level is frequently reduced in patients with extravascular haemolysis, and the Hp level cannot be used to determine whether the basic haemolytic process is intra- or extravascular. It should generally be accompanied by estimation of serum methaemalbumin, free plasma Hb, and urinary haemosiderin.
•Normal range (expressed as Hb-binding capacity): 30–250mg/dL.
•Incompatible blood transfusion.
•1% of the population have a genetic lack of Hps.
Note: it takes about 1 week after haemolysis has stopped for Hp levels to return to normal.
•Carcinoma, especially if bony 2°.
Rougemont A, Dumbo O, Bouvier M, et al. Hypohaptoglobinaemia as an epidemiological and clinical indicator for malaria. Results of two studies in a hyperendemic region in West Africa. Lancet 1988; 2: 709–12.
Two forms are found: pre-hepatic bilirubin (unconjugated) and bilirubin conjugated to glucuronic acid (conjugated). Generally, serum bilirubin levels are 17–50µmol/L in haemolysis (mainly unconjugated).
►Beware: serum bilirubin levels may be normal, even if haemolysis is present; a level of >85µmol/L suggests liver disease.
Serum bilirubin levels may be modestly ↑ (e.g. 20–30µmol/L) in dyserythropoietic disorders, such as vitamin B12 or folate deficiency, or myelodysplasia, due to ineffective erythropoiesis where RBCs are destroyed in the marrow before ever being released into the circulation.
Urobilinogen is the reduced form of urobilin, formed by bacterial action on bile pigments in the GIT. Faecal and urinary urobilinogen ↑ in haemolytic anaemias.
The most widely used and reliable test for detection of chronic intravascular haemolysis. Results from the presence of Hb in the glomerular filtrate.
Free Hb is released into the plasma during intravascular haemolysis. The Hb-binding proteins become saturated, resulting in the passage of haem-containing compounds into the urinary tract of which haemosiderin is the most readily detectable.
1.A clean-catch sample of urine is obtained from the patient.
2.The sample is spun down in a cytocentrifuge to obtain a cytospin preparation of urothelial cells.
3.Staining and rinsing with Perl’s reagent (Prussian blue) are performed on the glass slides.
4.Examine under the oil-immersion lens of a microscope.
5.Haemosiderin stains as blue dots within urothelial cells.
6.Ignore all excess stain and staining outside cells or in debris, all of which are common.
7.True +ve only when clear detection within urothelial squames is seen.
An iron-staining +ve control sample should be run alongside the test case to ensure the stain has worked satisfactorily. Haemosiderinuria may not be detected for up to 72h after the initial onset of intravascular haemolysis, so the test may miss haemolysis of very recent onset—repeat the test in 3–7 days if −ve. Conversely, haemosiderinuria may persist for some time after the haemolytic process has stopped. A repeat in 7 days should confirm.
(See Table 3.6.)
Table 3.6 Causes of haemosiderinuria
In health, Hb is contained within RBCs, but during intravascular haemolysis, excessive quantities of Hb may be released from ruptured RBCs. Normally Hps mop up free Hb. If there are insufficient Hps to cope with the free Hb, the kidneys clear the Hb, leading to haemoglobinuria. Some Hb may be broken down in the circulation to haem and globin; haem can bind to albumin, producing methaemalbumin (→ methaemalbuminaemia).
► The finding of free Hb in plasma is highly suggestive of intravascular haemolysis.
•Sample: sodium citrate (but discuss with the haematology laboratory before sending sample).
•Normal range: 10–40mg/L (up to 6mg/L).
•Pitfalls: any RBC damage occurring during blood sampling may result in an erroneously high reading. Great care must be taken during venepuncture.
(See Table 3.7.)
Table 3.7 Causes of increased plasma haemoglobin
| Mild ↑ (50–100mg/L) | Moderate ↑ (100–250mg/L) | Severe ↑ (>250mg/L) |
| Sickle/thalassaemia | AIHA | Incompatible blood transfusion |
| Haemoglobin C disease |
Crosby WH, Dameshek W. The significance of hemoglobinemia and associated hemosiderinuria, with particular reference to various types of hemolytic anemia. J Clin Lab Med 1951; 38: 829–41.
•Use: detection of methaemalbumin (seen after all Hps used up in a haemolytic process; usually implies haemolysis is predominantly intravascular).
This spectrophotometric test for methaemalbumin (which has a distinctive absorption band at 558nm) should be requested in patients with suspected intravascular haemolysis and may be abnormal in patients with significant extravascular (generally splenic) haemolysis. It should be accompanied by an estimation of the serum Hp level, free plasma Hb, and urinary haemosiderin.
•Sample: heparinized or clotted blood.
•Mismatched blood transfusion.
•G6PD deficiency with oxidative haemolysis.
Hoffbrand AV, Lewis SM, Tuddenham EGD (eds.). Postgraduate Haematology, 4th edn. Oxford: Butterworth-Heinemann, 2000.
Winstone NE. Methemalbumin in acute pancreatitis. Br J Surg 1965; 52: 804–8.
There are many inherited causes for haemolytic anaemia, which fall into three major groups, shown in Table 3.8.
Table 3.8 Inherited causes for haemolytic anaemiaa
| Mechanism | Example |
| Red cell membrane disorders | |
| Red cell enzyme disorders | |
| Hb disorders |
This is the best known inherited membrane abnormality leading to a reduced red cell lifespan and sometimes severe anaemia. Inheritance is usually autosomal dominant and there is often a +ve family history.
Usually autosomal dominant. Rarely causes symptomatic anaemia.
Autosomal recessive, often compound heterozygotes. Often severe haemolysis.
Heterozygotes asymptomatic.
Heterogeneous group with often severe haemolysis.
Immunophenotyping using the reagent EMA to bind to red cell transmembrane proteins. Altered fluorescence is seen with red cell membrane disorders.
Gel electrophoresis of membrane proteins. Only available in reference laboratories.
The test measures the ability of red cells to take up water before rupturing (lysing). This is determined by the volume:surface area ratio. Normal red cells can ↑ their volume by up to 70% before lysing (because they are disc-shaped and have the capacity to take in extra water easily). Spherocytic red cells have an ↑ volume:surface area ratio and are able to take up less water than normal red cells before lysing (they are spheres and, as such, they are ‘full’ already).
•Sample: EDTA (need a normal control sample sent at the same time).
(See Fig. 3.10.)
•RBCs are incubated in saline at various concentrations. This results in cell expansion and eventually rupture.
•Normal RBCs can withstand greater volume ↑ than spherocytic RBCs.
•A +ve result (confirming hereditary spherocytosis) seen when RBCs lyse in saline at near to isotonic concentration, i.e. 0.6–0.8g/dL (normal RBCs will simply show swelling with little lysis).
•Osmotic fragility is more marked in patients who have not undergone splenectomy and if the RBCs are incubated at 37°C for 24h before performing the test.
•There will be a +ve family history of hereditary spherocytosis in many cases.
•The blood film shows ↑↑ spherocytic RBCs.
•Anaemia, ↑ reticulocytes, ↑ LDH, unconjugated bilirubin, urinary urobilinogen with ↓ Hps.
►Beware: this test is not diagnostic of hereditary spherocytosis but will be +ve in any condition in which there are ↑ numbers of spherocytic red cells. Use this test in conjunction with a history, blood film, and family studies (hereditary spherocytosis is inherited as autosomal dominant, so one of the parents and some siblings should be affected).
Most testing laboratories use a combination of the above tests. No one screening test can detect all cases of hereditary spherocytosis.
Fig. 3.10 Test and control samples subjected to varying concentrations of saline. The broad grey band represents the normal range. The red cells in the test sample (from a patient with hereditary spherocytosis) lyse at higher concentrations of saline than that seen in the normal control, which suggests that hereditary spherocytosis is a likely diagnosis.
Bolton-Maggs PHB, Langer JC, Iolascon A, Tittensor P, King MJ. Guidelines for the diagnosis and management of hereditary spherocytosis – 2011 update. Br J Haematol 2012; 156: 37–49.
Parpart AK, Lorenz PB, et al. The osmotic resistance (fragility) of human red cells. J Clin Invest 1947; 26: 636–40.
Weatherall DJ, Provan AB. Red cells I: inherited anaemias. Lancet 2000; 355: 1169–75.
Numerous red cell enzymes are responsible for maintaining the integrity of the RBC in order to allow it to function efficiently in O2 delivery and CO2 removal. RBC enzyme defects lead to shortened RBC survival (i.e. haemolysis) and anaemia. Although there are numerous enzymopathies that may cause haemolysis, the most useful starting assays are for G6PD and pyruvate kinase (PK).
Of course, one should start by taking a detailed history from the patient, asking about previous haemolytic episodes, family history, ethnic origin, and possible drug toxicities.
•Sample: fresh EDTA or heparin. The enzymes are stable for 6 days at 4°C and 24h at 25°C.
•Methods: these are too numerous and complex to list here.
Essentially there are three methods for analysis of G6PD:
1.Brilliant cresyl blue decolorization test.
2.Methaemoglobin reduction test.
•Normal range: varies between laboratories (check with your local laboratory).
•Pitfalls: during a haemolytic episode in patients with G6PD deficiency, the oldest RBCs are destroyed first. Younger RBCs (and especially reticulocytes) have higher levels of the enzyme than older cells. It follows therefore that if the enzyme level is assayed during an acute episode, the G6PD level obtained may be falsely normal. This will rise further as reticulocytes pour into the peripheral blood, as happens during recovery from the acute attack. It is better to wait until the acute attack is over and the patient is in steady state.
Arya R, Layton DM, Bellingham AJ. Hereditary red cell enzymopathies. Blood Rev 1995; 9: 165–75.
Beutler E. The molecular biology of G6PD variants and other red cell enzyme defects. Annu Rev Med 1992; 43: 47–59.
World Health Organization Scientific Group (1967). Standardization of procedures for the study of glucose-6-phosphate dehydrogenase. Technical Report Series, No. 366. Geneva: World Health Organization.
There are two main classes of Hb abnormalities (see Table 3.9).
Table 3.9 Main classes of Hb abnormalities
| Abnormality | Example |
| Structural Hb variants | Sickle Hb, HbD, HbE |
| Imbalanced globin production | Thalassaemias (α, β, etc.) |
If the amino acid change results in an electrical charge difference, this may be detected by protein electrophoresis (separates proteins on the basis of charge). Investigation requires a full clinical history, FBC, blood film, and Hb electrophoresis.
β-thalassaemia is diagnosed from the blood indices, blood film, HbA2, and HbF levels. For α-thalassaemia, the investigation is more complex, requiring DNA analysis to detect α-globin deletions. Globin chain synthesis, which examines the ratio of α:β-globin production, is performed less with the advent of DNA-based methods.
Weatherall DJ, Provan AB. Red cells I: inherited anaemias. Lancet 2000; 355: 1169–75.
Electrophoresis is an electrical method for separating molecules on the basis of size (for DNA fragments) or overall electrical charge (for proteins). Hb electrophoresis allows the separation of different Hb, providing they have differing charges (Hb molecules with the same charge will move together on the gel and cannot be distinguished). The methods used take advantage of the fact that amino acid side chains on the globin molecules can be ionized. The net overall charge of a protein depends on the pH of the solution in which it is and the pKs of the amino acids (the pK is the pH at which half of the side chains are ionized).
•Cellulose acetate (at pH 8.6).
•High-performance liquid chromatography (HPLC).
Due to space limitations, each of these methods will be discussed only briefly. Other texts deal with this topic in considerable detail.
•Sample: peripheral blood EDTA.
This test is commonly performed in the diagnosis of abnormal Hb production (haemoglobinopathies or thalassaemia). Because some Hb have the same net charge, they will run together, e.g. HbS will run in the same band as HbD and HbG, and HbC will run with HbE. To resolve these bands, electrophoresis is next carried out at acid pH.
This is similar to cellulose acetate where Hb molecules are separated at an acid pH (pH 6.0) to separate out Hb that run together at alkaline pH.
This is a high-resolution method for separating different Hb molecules. The basic principle of the test relies on the fact that all proteins and amino acids have a pH at which their net charge is zero. This is termed the isoelectric point. At this pH, there is no net movement in the presence of an externally applied electric field. The Hb molecules are subjected to a pH gradient. This method has the advantage of high resolution but is more expensive than standard electrophoresis (see Fig. 3.11).
This chromatographic technique has been around for 20 years or more and is being increasingly used for analysis of Hb molecules. Hb molecules are passed through a matrix column and eluted from the column at varying times, during which their absorbance is measured. Detection of standard Hb variants is simple; the advantage of HPLC is that novel Hb variants can also be detected, and HPLC can separate proteins that cannot be resolved using other means. HPLC is more expensive than all the techniques mentioned above (see Figs 3.12 and 3.13).
•When the MCV is ↓↓, but Hb normal or slightly ↓.
•In patients from ethnic groups known to be associated with high levels of Hb disorder, e.g. sickle or thalassaemia.
1.Check the FBC and look at the MCV.
2.Is the MCV normal (>76fL)? If so, thalassaemia is unlikely.
3.Does the FBC show anything else? ↑ RCC with ↓ MCV and MCH are likely in thalassaemia.
4.Measure the HbA2; this is generally ↑ in β-thalassaemia trait (carrier).
7.Look at the distribution of HbF in RBCs (HbF is present in all RBCs in African hereditary persistence of fetal Hb (HPFH), but not present in all cells in carriers for ↓β-thalassaemia.
8.Assess the iron status (common cause of ↓ MCV—do not miss this!).
9.Look for RBC inclusions (e.g. H bodies in α-thalassaemia or Heinz bodies in unstable Hb disorders).
10.Carry out DNA analysis, examining both α- and β-globin genes.
Information Center for Sickle Cell and Thalassemic Disorders. Thalassemia. http://sickle.bwh.harvard.edu/menu_thal.html.
Sickle Hb is the result of a point mutation in the β-globin gene, resulting in a Glu → Val switch at position 6 of the β-globin protein. Sickle Hb (HbS) forms long filaments (tactoids), reducing its solubility when O2 tension is reduced. This forms the basis of the sickle solubility test (see Fig. 3.14).
Fig. 3.14 Blood film of homozygous sickle-cell anaemia (HbSS). Note the sickle-shaped (crescent) red cells.
The patient’s blood is mixed with sodium dithionite solution and left to stand. A +ve sickle sample should be used as a control. When the tubes are examined, a clear solution implies that there is no sickle Hb; a turbid solution confirms the presence of HbSS in the patient’s sample.
► A +ve result will be obtained for sickle carriers (HbAS) and sickle-cell homozygotes (HbSS). If a +ve result is obtained, Hb electrophoresis must be carried out to determine whether the patient is a carrier or has homozygous sickle-cell anaemia.
This is useful for prenatal diagnosis. The β-globin genes of the fetus are amplified using PCR; cells are obtained by amniocentesis or chorionic villus sampling (CVS) and digested with a bacterial restriction enzyme, e.g. Mst II. If the sickle mutation is present, no digestion will occur (the mutation removes the restriction site).
•Obtain blood from the neonate (e.g. heel prick) and in babies at risk of sickle or β-thalassaemia major (e.g. the mother has a gene for HbS, C, DPunjab, E, OArab, β- or ↓β-thalassaemia).
•Universal neonatal screening is generally used in areas where there is a high incidence of haemoglobinopathy.
OHCM 10e, p. 340.
Information Center for Sickle Cell and Thalassemic Disorders. Sickle cell disease. http://sickle.bwh.harvard.edu/menu_sickle.html.
Steinberg MH, Forget BG, Higgs DR, Weatherall DJ (eds.). Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. Cambridge: Cambridge University Press, 2001.
Normal adults have three types of Hb: HbA, HbA2, and HbF. HbA (α2β2) is the major Hb, and HbA2 is a minor adult Hb, which is very useful for the diagnosis of the β-thalassaemia trait. HbA2 levels are ↑ in the heterozygote (carrier state), and this is a specific test for this genotype. The test is carried out using a column chromatography method.
•β-thalassaemia trait (HbA2 level is ~3.9–6.5%).
Fetal Hb (HbF) makes up >50% of the total Hb at birth but ↓ to ~5% by 5 months of age (as γ chain production is replaced by β chains). HbF levels may be raised in some haemoglobinopathies.
•Hereditary persistence of HbF.
•Homozygous sickle-cell disease (HbSS).
•Sickle/β+ thalassaemia (some cases).
•Sickle/β0 thalassaemia (some cases).
•Multiple myeloma (uncommon and never measured).
HbH, consisting of a tetramer of β-globins (β4), is found in α-thalassaemia. The β chains form tetramers due to the relative lack of α-globins with which to pair. The demonstration of HbH allows the detection of the α-thalassaemia trait (either –α/–α or – –/αα) and HbH disease (– –/–α).
The HbH body test involves staining RBCs with brilliant cresyl blue; HbH bodies are seen as large, dark inclusions in the red cells.
►Note: the presence of HbH confirms α-thalassaemia, but the absence of HbH bodies does not exclude the diagnosis.
These are red cell inclusions made up of insoluble denatured globin protein. Heinz bodies are seen when RBCs are stained with methyl violet or new methylthioninium chloride (methylene blue) stain.
•Interpretation: Heinz bodies are seen close to the RBC membrane. These are normally removed by the spleen and are therefore more frequent following splenectomy.
•Chlorates, phenacetin, other drugs.
•G6PD and PK deficiencies, and other enzymopathies.
Sevitt S, Stone P, Jackson D, Baar S, Pollock A. Acute Heinz-body anaemia in burned patients. Lancet 1973; 2: 471–5.
Globin gene mutations may lead to amino acid substitutions that render the Hb molecule unstable, leading to haemolysis. Most mutations causing unstable Hb are autosomal dominant, and >80% affect the β chain. Affected individuals are heterozygotes. Heinz bodies in RBCs are intracellular Hb precipitates. Unstable Hb can be detected electrophoretically or by using the heat precipitation test, in which lysed RBCs are heated to 50°C for 1h.
•Interpretation: normal fresh haemolysates should be stable for 1h at 50°C. If there is an unstable Hb, a precipitate will be seen in the tube.
Although most haematology laboratories can diagnose β-thalassaemia trait and β-thalassaemia major, there are occasions when molecular tests are required, e.g. antenatal diagnosis where a couple are at risk of having a child with β-thalassaemia major or hydrops fetalis (absence of α-globin, usually lethal). In addition, the diagnosis of α-thalassaemia is difficult and requires DNA analysis, either using Southern blotting or PCR amplification of globin genes.
There are >100 β-globin mutations now known, but fortunately each population tends to have its own group of mutations (this avoids having to test for all known mutations). It is important that you include the ethnic group on the request form, since this will assist the laboratory who will then screen for mutations commonly found in the ethnic group of the patient. Details of these mutations can be found in the β- and δ-thalassemia repository.
The methods used are complex and outwith the scope of this small book.1 2–3
•This is amplification refractory mutation system PCR.
•Specific point mutations are known for the β-globin mutations.
•PCR primers are designed to bind with the mutated sequence.
•If the patient has the mutation, there will be PCR amplification.
•If the patient lacks the mutation, there is no binding of the primers to the patient’s DNA and no amplification.
•So a band on the gel means the mutation is present (and the reverse is true—if the band is absent, then that particular mutation is absent).
Other techniques, including reverse dot blots and DNA sequencing, are sometimes needed if ARMS PCR fails.
Whereas β-thalassaemia is usually the result of point mutations (single base changes), the α-thalassaemias are usually the result of deletions of chunks of DNA in the region of the α-globin genes. Southern blotting is useful in detecting deletions, since the DNA band sizes after digestion with restriction enzymes will differ to the wild type (i.e. normal).
This is based at the John Radcliffe Hospital in Oxford (UK). Difficult cases (e.g. α-thalassaemia) can be sent to this laboratory (after discussing the case first); they will perform α-globin gene analysis and send a detailed report containing the genotype of the patient. See end of the chapter for contact details ( Specialized haematology assays, pp. 330–331).
Determining the cause of haemolytic anaemia can be a complex process. Having excluded inherited disorders of Hb, RBC membrane, or enzymes, we are left with a diverse group of disorders with a common phenotype of ↑ RBC destruction (and ↓ RBC lifespan; see Table 3.10).
•Autoimmune (1°, or 2° to SLE or CLL).
•Alloimmune (e.g. transfusion reactions, haemolytic disease of the newborn (HDN)).
•Antibody can be warm (IgG) or cold (IgM usually).
•Thrombotic microangiopathies (TMAs)
There is little point investigating the cause of haemolytic anaemia until you have shown that haemolysis is actually occurring.
•Spherocytes (suggests warm antibody; also present in hereditary spherocytosis).
•↑ WBC, e.g. might suggest an underlying lymphoproliferative disorder such as CLL.
•RBC fragments (suggests physical damage to the RBC, e.g. microangiopathic haemolytic anaemia (MAHA), TTP/HUS, burns, March haemoglobinuria, mechanical heart valves).
•Infections, e.g. Clostridium, Bartonella, Babesia.
•IgG or IgG + complement (C3d) on RBC.
•DAT is usually +ve in immune-mediated haemolysis.
•Renal function (often abnormal in TTP/HUS).
•Coagulation screen (DIC with RBC fragmentation).
•LFTs (abnormal in Zieve’s syndrome).
•Cold agglutinins: IgM, usually against I or i proteins, RBC membrane proteins.
•Immunophenotype if suspect PNH.
Table 3.10 Acquired haemolytic anaemias: mechanisms
PNH is a rare acquired red cell membrane disorder. Loss of glycosyl phosphatidyl inositol (GPI)-linked surface proteins make the red cells vulnerable to complement-mediated lysis, causing episodes of marked intravascular haemolysis, with free Hb in the urine (haemoglobinuria). Immunophenotyping shows cells to be deficient in the GPI-anchored proteins CD55 and CD59 (erythrocytes) and CD16, CD24, and FLAER (granulocytes),
The technique of immunophenotyping is described later in this chapter.
This is a test of 1° haemostasis, and mainly of platelet function in vivo, rather than a laboratory test. You will generally need to arrange this test through the haematology department who will carry out the test for you.
A disposable spring-loaded blade is used to make two incisions of fixed depth into the skin of the forearm, whilst a sphygmomanometer is inflated to 40mmHg. Blood from the incisions is mopped up using circular filter paper (care needs to be taken to avoid disturbing the clot that forms on the cut surface).
•Normal range: up to 7min (varies, depending on the method used; >9min is abnormal). Longer in ♀.
•Uses: previously felt to be the best screen for acquired or congenital functional or structural platelet disorders. If bleeding time is normal and history −ve (i.e. no major bleeding problems in the past), this excludes an underlying platelet disorder.
► Do not carry out bleeding time if the platelet count is <100 × 109/L (will be prolonged). Aspirin will interfere with the test—ask patients to stop aspirin 7 days before the test is carried out.
•Platelet function defect (acquired, e.g. aspirin, paraprotein, MDS).
•von Willebrand’s disease (vWD).
•Vascular abnormalities, e.g. Ehlers–Danlos.
•Occasionally low factor V or XI.
Highly operator-dependent, with low reproducibility. Because of this, the test is seldom used now.
Mielke CH Jr. Aspirin prolongation of the template bleeding time: influence of venostasis and direction of incision. Blood 1982; 60: 1139–42.
Parkin JD, Smith IL. Sex and bleeding time. Thromb Haemost 1985; 54: 731.
This tests the extrinsic coagulation pathway and is useful for detecting coagulation deficiencies, liver disease, and DIC. The prothrombin time (PT) is also the main monitor for coumarin therapy (e.g. warfarin), expressed as a ratio—the international normalized ratio (INR). The test measures the clotting time of plasma in the presence of a tissue extract, e.g. brain (thromboplastin). The test measures prothrombin, but also factors V, VII, and X (see Fig. 3.15).
•Oral anticoagulation therapy (vitamin K antagonists).
•Fibrinogen deficiency (factor I).
•Prothrombin deficiency (factor II).
•Deficiency of factors V, VII, or X (in V or X deficiency, the activated partial thromboplastin time (APTT) will be ↑).
•Liver disease, especially obstructive.
Fig. 3.15 Coagulation cascade showing the factors assayed using the various clotting tests.
Modified from Provan D, et al. Oxford Handbook of Clinical Haematology, 2nd edn. Oxford: Oxford University Press, 2004.
Provan D, Singer CRJ, Baglin T, Lilleyman J.Oxford Handbook of Clinical Haematology, 2nd edn. Oxford: Oxford University Press, 2004.
Other terms: somewhat confusingly, the APTT may be called kaolin cephalin clotting time (KCCT) or partial thromboplastin time with kaolin (PTTK).
This is a test of the intrinsic coagulation system and depends on contact factors + factors VIII and IX, and reactions with factors X, V, II, and I. The APTT is sensitive to circulating anticoagulants (e.g. lupus anticoagulant) and heparin.
2.Screening for haemophilia A and B (VIII and IX deficiencies, respectively).
3.Screening for coagulation inhibitors.
•Normal range: 26.0–33.5s (often expressed as activated partial thromboplastin time ratio (APTR)).
•Modest ↑ in patients taking oral anticoagulants.
The APTT will be long if there is an inhibitor, such as the lupus anticoagulant, present. This can be determined by mixing the patient’s plasma with an equal volume of normal control plasma and repeating the APTT. This is known as a 50:50 mix. If the APTT is long because of an inhibitor, it will not fully correct when normal plasma is added. However, if the APTT is long because of a deficiency, it will be corrected with the normal plasma.
Denson KW. Thromboplastin—sensitivity, precision and other characteristics. Clin Lab Haematol 1988; 10: 315–28.
Lab Tests Online (2012). Coagulation cascase. http://www.labtestsonline.org/understanding/analytes/coag_cascade/coagulation_cascade.html.
Turi DC, Peerschke EI. Sensitivity of three activated partial thromboplastin time reagents to coagulation factor deficiencies. Am J Clin Pathol 1986; 85: 43–9.
van den Besselaar AM, Meeuwisse-Braun J, Jansen-Grüter R, Bertina RM. Monitoring heparin therapy by the activated partial thromboplastin time—the effect of pre-analytical conditions. Thromb Haemost 1987; 57: 226–31.
This is affected by the concentration of factor I (fibrinogen) and the presence of fibrin or fibrinogen degradation products and heparin.
•↑ FDPs/cross-linked fibrin degradation products (XDPs)/D-dimers.
•Dysfibrinogenaemia (inherited; mutation in the fibrinogen gene leads to amino acid change and non-functional fibrinogen).
Note: (*) if suspected, check reptilase time, similar to thrombin clotting time (TCT), but not affected by heparin.
D-dimers are produced during polymerization of fibrinogen as it forms fibrin. Measurement of D-dimer levels is more specific for this process than the older FDP test and is now being used to detect the presence of DIC and other coagulation disorders. The test measures fibrin lysis by plasmin and is a sensitive indicator of coagulation activation (e.g. such as that seen in DIC). The assay uses an MoAb specific for D-dimers; it will not cross-react with fibrinogen or fibrin.
•Sample: citrate (clotting screen bottle).
(See Table 3.11.)
Table 3.11 Summary of clotting tests in a variety of disorders
| PT | APTT | TCT | Platelets | Diagnosis |
| N | N | N | N | Platelet function defect, XIII deficiency, normal |
| ↑ | N | N | N | VII deficiency, early oral anticoagulation |
| N | ↑ | N | N | VIIIC/IX/XI/XII deficiencies, vWD, circulating anticoagulant, e.g. lupus |
| ↑ | ↑ | N | N | Vitamin K deficiency, oral anticoagulant, V/VII/X/II deficiencies |
| ↑ | ↑ | ↑ | N | Heparin, liver disease, fibrinogen deficiency |
| N | N | N | ↓ | Thrombocytopenia (any cause) |
| ↑ | ↑ | N | Low | Massive transfusion, liver disease |
| ↑ | ↑ | ↑ | Low | DIC, acute liver disease |
Modified from Dacie & Lewis Practical Haematology, 8th edn. Edinburgh: Churchill Livingstone, 1995.
DIC is a medical and haematological emergency. It may be seen in a variety of situations and is characterized by generalized bruising and bleeding, usually from venepuncture sites, post-operatively, and spontaneously.
Diagnosis requires FBC, clotting screen, and evidence of rapid consumption of fibrinogen. Classic (acute) DIC, where the test results fit the bill, is easy to spot. The situation may be more subtle and you are strongly advised to discuss the case with a haematology registrar or consultant if you are in any doubt about the diagnosis of DIC.
| ►► Laboratory diagnosis | |
| FBC | |
| PT | ↑ in moderately severe DIC |
| APTT | Usually ↑ |
| Fibrinogen | ↓ (falling levels significant—but remember this is an acute phase protein, so levels may be normal, even in florid DIC) |
| D-dimers | ↑ |
(See Table 3.12.)
Table 3.12 Conditions associated with DIC
Levi MM (2016). Disseminated intravascular coagulation. http://www.emedicine.com/emerg/topic150.htm.
These are specialized tests carried out by the coagulation laboratory for the investigation of patients with suspected platelet dysfunction. Because of their complexity, platelet function tests will not be described in detail here.
Patients generally present with bleeding or bruising problems and have had normal coagulation results. Because of the labour-intensive nature and cost of these assays, you will need to arrange these tests after discussion with your local haematology medical staff.
•Sample: blood collection needs to be optimal with non-traumatic venepuncture, rapid transport to the laboratory with storage at room temperature and testing within a maximum of 2–3h.
Normal range 150–400 × 109/L. Adequate function is maintained, even when the count is <0.5 normal level but progressively deteriorates as it drops. With platelet counts <20 × 109/L, there is usually easy bruising and petechial haemorrhages (although more serious bleeding can occur).
Large platelets are biochemically more active; ↑ mean platelet volume (MPV >6.5) is associated with less bleeding in patients with severe thrombocytopenia. Altered platelet size is seen in inherited platelet disorders.
Adhesion to glass beads now rarely performed in routine laboratory practice, but potentially useful in vWD diagnosis.
Most useful of the special tests; is performed on a fresh sample using an aggregometer.
•Adenosine 5-diphosphate (ADP) at low and high concentrations. Induces two aggregation waves: the 1° wave may disaggregate at low concentrations of ADP; the second is irreversible.
•Collagen has a short lag phase, followed by a single wave, and is particularly affected by aspirin.
•Ristocetin-induced platelet aggregation (RIPA) is carried out at high (1.2mg/mL) and lower concentrations and is mainly used to diagnose vWD.
•Adrenaline (epinephrine), not uncommonly reduced in normal people.
Enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) are used to measure the granule proteins β-thromboglobulin (β-TG) and heparin neutralizing activity (HNA). These are sensitive markers of platelet hyper-reactivity and beyond the scope of the routine laboratory.
Their main role is in diagnosis of inherited platelet functional defects. In acquired platelet dysfunction 2° to causes such as renal and hepatic disease, DIC, and macroglobulinaemia, platelet function is rarely tested.
Yardumian DA, Mackie IJ, Machin SJ. Laboratory investigation of platelet function: a review of methodology. J Clin Pathol 1986; 39: 701–12.
Thrombophilia describes acquired or inherited disorders that predispose to arterial or venous thromboembolism (VTE). Thrombophilia should be considered in young patients who have apparent strong family history of VTE.
•Causes: Recurrent thrombosis, p. 92.
•Arterial thrombosis, consider antiphospholipid antibody and lupus anticoagulant testing.
•Patients <40 years from thrombosis-prone families.
•Family history of thrombosis with high-risk thrombophilia in first-degree relative.
•Unusual site, e.g. mesenteric, portal vein thrombosis.
•Children with purpura fulminans.
•Recurrent miscarriage (three or more).
•VTE in pregnancy and the OCP.
•Exclude medical causes (check ESR, LFTs, AIP, fasting lipids).
•FBC (exclude thrombocytosis).
•Clotting screen for acquired defects (PT, APTT, LA/anticardiolipin antibody (ACL), ↑ fibrinogen).
•Screen for inherited thrombophilia:
•First-line protein C (PC), protein S (PS), antithrombin (AT), activated protein C resistance (APCR).
•Check for the presence of the factor V Leiden mutation in APCR +ve patients (DNA analysis).
•Consider testing plasminogen, fibrinogen, homocysteine levels, prothrombin variant.
•DNA analysis for prothrombin gene mutation.
Thrombophilia investigations are time-consuming and expensive, and you should discuss with the local haematology medical or laboratory staff before sending samples. Note: some thrombophilia tests cannot be carried out in the ‘acute’ phase of a VTE event or whilst the patient is taking an anticoagulant.
Baglin T, Gray E, Greaves M, et al. Clinical guidelines for testing for heritable thrombophilia. Br J Haematol 2010; 149: 209–20.
Cattaneo M, Monzani ML, Martinelli I, Falcon CR, Mannucci PM. Interrelation of hyperhomocyst(e)inemia, factor V Leiden, and risk of future venous thromboembolism. Circulation 1998; 97: 295–6.
Chong LY, Fenu E, Stansby G, Hodgkinson S. Management of venous thromboembolic diseases and the role of thrombophilia testing: summary of NICE guidance. BMJ 2012; 344: e3979.
Dahlback B. Resistance to activated protein C as risk factor for thrombosis: molecular mechanisms, laboratory investigation, and clinical management. Semin Hematol 1997; 34: 217–34.
Lane DA, Mannucci PM, Bauer KA, et al. Inherited thrombophilia: Part 1. Thromb Haemost 1996; 76: 651–62.
Lane DA, Mannucci PM, Bauer KA, et al. Inherited thrombophilia: Part 2. Thromb Haemost 1996; 76: 824–34.
These proteins are the body’s natural anticoagulants; hence, deficiencies may lead to thromboembolic disease.
Used to be called ATIII, but there was never an ATI or ATII, so now abbreviated to AT. A useful measure in thrombophilia screening since low levels of AT are found in 4.5% of patients with unexplained VTE.
•Hereditary (40–60% normal level), autosomal dominant.
•Third trimester of pregnancy.
•Reduced levels predispose to VTE. Individuals with 30–60% normal level may suffer recurrent thrombosis.
•Inherited (autosomal dominant).
•Oral anticoagulants, e.g. warfarin.
•Similar to PS; autosomal dominant inheritance in genetic cases.
This is a key investigation in haematology. It may be diagnostic in the follow-up of abnormal peripheral blood findings and is an important staging procedure in defining the extent of disease, e.g. lymphomas. It is a helpful investigative procedure in unexplained anaemia, splenomegaly, or selected cases of PUO.
•Preferred sites: the posterior iliac crest is the usual site (allows an aspirate and a biopsy to be obtained). The sternum is suitable only for marrow aspiration and is not a test for the squeamish.
•Qualitative and semi-qualitative analysis of haematopoiesis.
•Assessment of iron stores (if Perls’ iron stain used).
•Smears for cytochemistry (helps in the diagnosis of leukaemias).
•Chromosomal (cytogenetic) analysis.
•Immunophenotype studies using MoAbs.
•Identification and classification of abnormal cells.
•Immunohistochemistry on infiltrates.
None, other than physical limitations, e.g. pain or restricted mobility. Avoid sites of previous radiotherapy (inevitably grossly hypocellular and not representative). (See Table 3.13.)
1.BM aspiration may be performed under local anaesthetic (LAn) alone, but short-acting IV sedative (e.g. midazolam) is preferred when a trephine biopsy is performed. General anaesthetic used in children.
2.Place the patient in the (left) lateral position, or use the right side if s/he cannot lie on the left side.
3.Infiltrate the skin and periosteum over the posterior iliac spine with LAn.
4.Make a small cutaneous incision before introducing the aspirating needle, which should penetrate the marrow cortex 3–10mm before removal of the trocar.
5.Aspirate no more than 0.5–1mL of marrow initially (to avoid dilution of the sample with blood).
6.Make smears promptly (► the sample clots rapidly!).
7.If further samples are needed, e.g. for immunophenotyping, cytogenetics, etc., these can be aspirated after making initial slides.
8.For trephine biopsy, use an Islam or Jamshidi needle.
9.Advance the needle through the same puncture site to penetrate the cortex.
10.Remove the trocar and, using firm hand pressure, rotate the needle clockwise and advance as far as possible.
11.Remove the needle by gentle anticlockwise rotation.
12.Following the procedure, apply simple pressure dressings.
13.Minor discomfort at the location may be dealt with by simple analgesia such as paracetamol.
Table 3.13 Tests carried out on bone marrow
These staining methods have been around for many years (for decades, they were all that was available) but remain extremely useful in the diagnosis and classification of leukaemias. Modern technologies, such as flow cytometry and nucleic acid analysis, have refined leukaemia and lymphoma diagnosis, but the examination of well-stained cytochemistry BM smears remains the cornerstone of good haematology practice.
After performing a BM aspirate and spreading the material onto glass slides, the air-dried, unfixed microscope slides are passed to the cytochemistry laboratory that will fix and stain the slides according to the likely diagnosis (e.g. stains for AML differ to those for ALL; see Tables 3.14 and 3.15). Positive results with particular stains will point to a specific diagnosis. This will then be augmented by flow cytometric or molecular assays (see Fig. 3.16).
Fig. 3.16 AML marrow showing myeloblasts (leukaemic cells) and an Auer rod in one cell. Auer rods are pathognomonic of AML, since they do not occur in any other disorder.
Table 3.14 Cytochemical stains
| Cytochemical stain | Substrate/cell |
| Myeloperoxidase (MPO) | Lysosomal enzyme found in neutrophils and monocytes |
| Sudan black (SB) | Phospholipids in neutrophil granules |
| Chloroacetate esterase | Stain-specific esterase in granulocytes and mast cells. Makes it easier to diagnose AML M4 subtype |
| α-naphthyl acetate esterase (ANAE) | Esterase stain, useful for diagnosis of AML subtypes |
| Acid phosphatase | Enzyme found in many different WBCs. Useful for T-cell malignancies |
| Periodic acid–Schiff (PAS) | Detects glycogen in cells. Granulocytes have diffuse staining, whereas lymphocytes staining is much coarser |
Table 3.15 Cytochemical stains in acute leukaemia
| Acute lymphoblastic leukaemia | Acute myeloid leukaemia | ||||
| B lineage | T lineage | M1–3 | M4–5 | M6–7 | |
| MPO | − | − | +/++ | + | − |
| SB | − | − | +/++ | + | − |
| Chloroacetate esterase | − | − | −/++ | − | − |
| ANAE | − | − | − | ++ | − |
| Acid phosphatase | − | + (focal) | − | + (diffuse) | + (focal) |
| PAS | + (blocks) | − | − | + (fine granular) | + |
+, positive; ++, strongly positive; −, negative.
From Hoffbrand AV, Lewis SM, Tuddenham EGD.Postgraduate haematology, fourth edition, Oxford: Butterworth-Heinemann, 2000.
Cytochemistry. https://clinicalgate.com/cytochemistry/
This is a cytochemical stain used to demonstrate the presence and quantity of the neutrophil enzyme ALP. Historically, the NAP score was of value in differentiating ‘reactive’ states from myeloproliferative disorders such as CML, PRV, etc.—now more often it features in examination multiple choice questions! (Note: sometimes termed leucocyte alkaline phosphatase, LAP.)
Best performed on fresh blood films, made without the use of anticoagulant. EDTA samples may be used but are less satisfactory. The film should be made, air-dried, fixed, and then stained—all within 30min. Positive NAP activity is indicated by the presence of bright blue granules in the neutrophil cytoplasm (the nucleus is stained red; see Fig. 3.17).
•Scoring: films are scored from 0 to 4 on the basis of stain intensity:
•2: +ve, few to moderate numbers of granules.
Fig. 3.17 NAP-stained blood film showing positively stained neutrophils. Red cells do not take up the stain.
(See Table 3.16.)
| High NAP score | Low NAP score |
The NAP score is affected by corticosteroids, oestrogens, and pregnancy (↑ NAP). In Hodgkin’s disease, the NAP score offers no advantage over simpler tests, such as ESR, for assessment of disease activity. Occasionally of value in a patient with aplastic anaemia who is developing PNH—the NAP score is seen to fall (both of these are very rare disorders). NAP score has been replaced in most hospitals by flow cytometry and other methods.
Lewis SM, Bain BJ, Bates I (eds.). Dacie & Lewis Practical Haematology, 9th edn, Edinburgh: Churchill Livingstone, 2001.
Due to space limitation, it is inappropriate to go into major details about the investigations used in transfusion medicine. However, we have provided the more important tests in current use which include:
•Blood group and antibody screen.
•Cross-match (compatibility test).
•Antiplatelet and antineutrophil antibody testing.
Each year, patients are transfused with the wrong blood. In 2015, seven patients were given ABO-incompatible blood, whilst 280 patients were given incorrect blood components (not just ABO-incompatible) in 2015.4 Note that reporting is currently voluntary and very likely underestimates actual incidents. However, with 2.5 million blood components transfused annually, it is a small percentage overall. Pulmonary complications, particularly transfusion-associated circulatory overload (TACO), are a major cause of morbidity and death. However, it is also clear that delay in appropriate transfusion also contributes to mortality.
A common error is clerical and generally involves the cross-matched sample being taken from the wrong patient, and so the compatibility test is performed on the wrong sample. Occasionally, the staff carrying out the transfusion connect the blood up to the wrong patient. In any event, the result varies from no symptoms to shock and possible death.
•First, ask yourself, ‘Does this patient really need to be transfused with blood or blood products (e.g. fresh frozen plasma (FFP), platelets, etc.)?’ For example, a post-operative patient who is asymptomatic with Hb of 9g/dL probably does not require red cell transfusion. Use clinical judgement in helping decide whether or not to proceed with transfusion.
•Before taking the blood sample, check that you are taking blood from the correct patient—ask for his/her name and check the identity bracelet.
•Label the patient’s blood bottle at the bedside (i.e. no prelabelling of bottles). Many transfusion laboratories insist on 1, 2, 5, 6, and 7, and either 3 or 4 from:
•Ensure details on the form match those on the bottle.
•Complete the request form properly:
•State what is required (e.g. 2U of packed cells, etc.).
•Detail any previous transfusions, reactions, antibodies (if known).
•Let the laboratory know when you want the blood or blood product.
►► Adhesive patient labels are fine for forms but are not suitable for specimen bottles, and they are usually not accepted by transfusion laboratories. Transfusion specimens should be labelled by hand—at the bedside.
The TACO checklist should be used to identify those at risk. Defer transfusion, if safe, pending assessment or treatment of risk factor. Single unit transfusion, followed by a review, is preferable.
The bedside checklist should be used to:
•Confirm +ve patient identification.
•Check identification of the component against the patient’s wristband.
•Check for specific requirements.
Remember many people die annually because they are transfused with the wrong blood. In most cases, clerical error is to blame—people have filled out bottles in advance and failed to check patient identity.
Estcourt LJ, Birchall J, Allard S, et al.; British Committee for Standards in Haematology. Guidelines for the use of platelet transfusions. Br J Haematol 2017; 176: 365–94.
Hunt BJ, Allard S, Keeling D, Norfolk D, Stanworth SJ, Pendry K; British Committee for Standards in Haematology. A practical guideline for the haematological management of major haemorrhage. Br J Haematol 2015; 170: 788–803.
Joint United Kingdom (UK) Blood Transfusion and Tissue Transplantation Services Professional Advisory Committee (JPAC). http://www.transfusionguidelines.org.uk/.
McClelland B (2001). Handbook of transfusion medicine, second edition. London: HMSO.
NHS Blood and Transplant. http://www.blood.co.uk/.
Scottish National Blood Transfusion Service. http://www.scotblood.co.uk/.
(See Table 3.17.)
Rapid fever, chill, rigor, hyper- or hypotension, collapse, flushing, urticaria, or respiratory distress at the start of transfusion indicate transfusion should be stopped and resuscitation initiated (suggests a severe or life-threatening acute transfusion reaction).
If the temperature rises to above 39°C or >2°C from baseline, with other signs/symptoms, consider bacterial contamination and monitor the patient carefully. Investigate as appropriate.
•If an isolated temperature of >38°C and a 1–2°C rise with no symptoms, or rash only, providing the patient is not acutely unwell, continue the infusion. Fever often due to antibodies against WBCs (or to cytokines in platelet packs).
Intravascular haemolysis (→ haemoglobinaemia and haemoglobinuria). Usually due to anti-A or anti-B antibodies (in ABO-mismatched transfusion). Symptoms occur in minutes/hours. May be fatal.
Table 3.17 Transfusion reactions
| Symptoms | Signs |
| Patient restless/agitated | Fever |
| Flushing | Hypotension |
| Anxiety | Oozing from wounds or venepuncture sites |
| Chills | Haemoglobinaemia |
| Nausea and vomiting | Haemoglobinuria |
| Pain at venepuncture site | |
| Abdominal, flank, or chest pain | |
| Diarrhoea |
If predominantly extravascular, may only suffer chills/fever 1h after starting transfusion—commonly due to anti-D. ARF is not a feature.
Complement (C3a, C4a, C5a) release into recipient plasma → smooth muscle contraction. May develop DIC or oliguria (10% of cases) due to profound hypotension.
•Stop blood transfusion immediately.
•Replace the giving set; keep the IV access open with 0.9% saline.
•Check the patient identity against the donor unit.
•Insert a urinary catheter and monitor the urine output.
•Give fluids (IV colloids) to maintain urine output >1.5mL/kg/h.
•If urine output <1.5mL/kg/h, insert a CVP line and give a fluid challenge.
•If urine output <1.5mL/kg/h and CVP adequate, give furosemide 80–120mg.
•If urine output still <1.5mL/kg/h, consult senior medical staff for advice.
•Contact the blood transfusion laboratory before sending back the blood pack and for advice on blood samples required for further investigation ( Urgent investigations below).
Your local blood transfusion department will have specific guidelines to help you with the management of an acute reaction. The following guide lists the samples commonly required to establish the cause and severity of a transfusion reaction (see Box 3.1). If you are uncertain about the laboratory procedure or management of a patient who appears to have suffered a severe reaction, you must notify your hospital’s haematology medical staff who will provide advice.
►► Delays may threaten the patient’s life.
Box 3.1 Investigation of transfusion reaction
1.Check the compatibility label of the blood unit matches with the patient’s identity band, forms, and case notes.
2.If mistake found, tell the blood bank urgently—the unit of blood intended for your patient may be transfused to another patient.
•Microbiology: blood cultures.
4.Check urinalysis and monitor urine output.
5.Do ECG and check for evidence of ↑ [K+].
6.Arrange repeat coagulation screens and biochemistry 2- to 4-hourly.
Seen in 0.5–1.0% of blood transfusions. Mainly due to anti-HLA (human leucocyte antigen) antibodies in recipient serum or granulocyte-specific antibodies (e.g. sensitization during pregnancy or previous blood transfusion).
Occurs in patients immunized through previous pregnancies or transfusions. Antibody weak (so not detected at pre-transfusion stage). 2° immune response occurs—antibody titre ↑.
•Occur 7–10 days after blood transfusion.
•Fever, anaemia, and jaundice.
•Discuss with the transfusion laboratory staff.
•Check DAT and repeat compatibility tests.
•Transfuse the patient with freshly cross-matched blood.
Kardon EM (2016). Transfusion reactions in emergency medicine. http://www.emedicine.com/emerg/topic603.htm.
Uncommon, but potentially fatal, adverse effect of blood transfusion (affects red cells and blood products, e.g. platelet concentrates). Implicated organisms include Gram −ve bacteria, including Pseudomonas, Yersinia, and Flavobacterium.
Include fever, skin flushing, rigors, abdominal pain, DIC, ARF, shock, and possible cardiac arrest.
As per Immediate transfusion reaction ( p. 304):
•IV broad-spectrum antibiotics if bacterial contamination suspected.
The old term is Coombs’ test. DAT detects antibodies or complement, or both, on the RBC surface, and the indirect antiglobulin test (IAT) detects the presence of antibodies in serum. A useful investigation when investigating haemolytic anaemia.
•Lymphoproliferative disorders, e.g. CLL.
•Drug-induced haemolysis (e.g. methyldopa, levodopa).
•Haemolytic disease of the fetus and newborn (HDFN), e.g. rhesus (Rh) HDN.
Note: as with many tests in medicine, things are never entirely black or white—a +ve DAT does not necessarily imply that haemolysis is actively occurring and a −ve DAT does not exclude haemolysis.
Coombs RRA. A new test for the detection of weak and ‘incomplete’ Rh agglutinins. Br J Exp Pathol 1945; 26: 255–66.
Kelton JG. Impaired reticuloendothelial function in patients treated with methyldopa. N Engl J Med 1985; 313: 596–600.
To determine whether fetal red cells have entered the maternal circulation and, if so, the volume of such fetal cells.
If an Rh (D) −ve mother has a baby that is Rh (D) +ve, she may develop antibodies (maternal anti-D) against fetal red cells. This may result in fetal red cell destruction termed rhesus haemolytic disease of the newborn, a serious haemolytic disorder that is seen less today due to a greater understanding of the underlying mechanism and our ability to prevent it. Sensitization to the fetal red cells occurs when fetal RBCs enter the maternal circulation, e.g. at birth or through obstetric manipulations, e.g. amniocentesis, previous pregnancies, etc.
Fetal RBCs in the mother’s circulation can be detected and quantified (in mL) using the Kleihauer test, which exploits the resistance of fetal red cells to acid elution (acid washes adult Hb out of the mother’s red cells, but fetal RBCs contain HbF, which is not washed out). The Kleihauer test should be performed on all Rh (D) −ve women who deliver an Rh (D) +ve infant.
Fetal cells appear as darkly staining cells against a background of ghosts (these are the maternal red cells). An estimate of the required dose of anti-D can be made from the number of fetal cells in a low-power field.
•Sample: maternal peripheral blood EDTA.
Basically, a calculation is made by the laboratory staff, based on the number of fetal RBCs seen in the Kleihauer film. The actual calculation is:
For example, if there are 1% of fetal RBCs in maternal circulation:
(* where 1800 is the maternal red cell volume)
A 4mL bleed (i.e. 4mL of fetal RBCs) requires 500IU of anti-D given IM to the mother, with a further 250IU of anti-D for each additional mL of fetal RBCs.
The laboratory carrying out the Kleihauer test will tell you the volume of fetal RBCs detected, since they will count the cells and do the calculation for you. If the total is >2mL of fetomaternal haemorrhage, the maternal sample is sent for FMH confirmation by flow cytometry. After this, you will need to calculate the dose of anti-D to give the mother, but if you are unsure, either discuss with the haematology medical staff or contact your local transfusion centre.
Chanarin I (ed.). Laboratory Haematology: An Account of Laboratory Techniques. Edinburgh: Churchill Livingstone, 1989.
Epo is the hormone produced largely by the kidney that drives red cell production. The typical anaemia found in renal disease is a result of failure of Epo production. Epo assays are of value in renal medicine and haematology. For example, in the assessment of polycythaemic states, an ↑ Epo level may be appropriate (e.g. in hypoxia where the body is attempting to ↑ O2 availability to tissues) or inappropriate (e.g. some tumours). The Epo assay is carried out using an RIA method and is not available in all haematology laboratories (may need to be sent to another hospital or laboratory).
•Normal range: 35–25mU/mL, steady-state level, no anaemia. May rise to 10,000mU/mL in hypoxia or anaemia.
•Cyanotic heart disease (e.g. right → left shunts).
•Cerebellar haemangioblastoma.
•RhA and other chronic inflammatory diseases.
Cotes PM, Doré CJ, Yin JA, et al. The use of immunoreactive erythropoietin in the elucidation of polycythemia. N Engl J Med 1986; 315: 283–7.
Immunohaematology is the study of the effects of the immune system on the blood and its components. This includes red cells, white cells, platelets, and coagulation proteins.
These tests are usually requested by the haematology department for patients with either thrombocytopenia or neutropenia, respectively. These assays are used to detect the presence of specific antibodies against platelet or neutrophil antigens on the cell surface.
Antibodies may be alloantibodies (e.g. antibodies produced by the mother against fetal antigens) or autoantibodies, which are antibodies produced by the patient against his/her own antigens (see Table 3.18).
Generally platelet immunofluorescence tests (PIFTs) or monoclonal antibody immobilization of platelet antigens (MAIPA) are used. These are useful for detecting even weak antibodies or where there are only a few antigenic sites per cell.
Table 3.18 Disorders with neutrophil-specific allo- and autoantibodies
Disorders with neutrophil-specific alloantibodies •Neonatal alloimmune neutropenia |
Disorders with neutrophil-specific autoantibodies •Evans’ syndrome (AIHA + ↓ platelets) |
Elegant though these tests are, they are actually not useful in clinical practice for the diagnosis of neutropenia or thrombocytopenia where the cause is autoimmune, since these are largely clinical diagnoses. (Platelet-associated IgG or IgM may be high in autoimmune thrombocytopenia. However, it may also be high in non-immune causes of thrombocytopenia.) Where these tests are of value is in the neonatal setting where the neonate has low platelets or neutrophils.
Roitt I.Essential Immunology, 10th edn. Oxford: Blackwell Science, 2001.
This describes the identification and counting of cell types using powerful MoAbs specific for cell surface proteins.
(See Table 3.19.)
•Diagnosis and classification of leukaemias and lymphomas.
•Assessment of cellular DNA content and synthetic activity.
•Determination of lymphocyte subsets, e.g. CD4+ T cells in HIV infection.
•Allows identification of prognostic groups.
•Monitoring of minimal residual disease (MRD, the lowest level of malignancy that can be detected using standard techniques).
Cell surface proteins are denoted according to their cluster differentiation (CD) number. Most cells will express many different proteins, and the pattern of expression allows cellular characterization. MoAbs recognize specific target antigens on cells. Using a panel of different antibodies, an immunophenotypic profile of a sample is determined. Immunophenotyping is used in conjunction with standard morphological analysis of blood and marrow cells. The antibodies are labelled with fluorescent markers, and binding to cell proteins is detected by laser. For each analysis, thousands of cells are assessed individually and rapidly. Some antibodies can detect antigens inside cells.
These are so-called because they are derived from single B lymphocyte cell lines and have identical antigen-binding domains (idiotypes). It is easy to generate large quantities of MoAbs for diagnostic use.
•Cell populations from, e.g. peripheral blood or BM samples are incubated with a panel of MoAbs, e.g. anti-CD4, anti-CD34, which are directly or indirectly bound to a fluorescent marker antibody.
•Sample is passed through a fluorescence-activated cell sorter (FACS) machine.
•FACS instruments assign cells to a graphical plot by virtue of cell size and granularity detected as forward and side light scatter by the laser.
•Allows subpopulations of cells, e.g. mononuclear cells, in blood samples to be selected.
•The reactivity of this cell subpopulation to the MoAb panel can then be determined by fluorescence for each MoAb.
•A typical result for a CD4 T-lymphocyte population is shown: CD3, CD4 +ve; CD8, CD13, CD34, CD19 −ve.
•AML: CD13+, CD33+, ± CD34, ± CD14 +ve.
•B-ALL: CD10, CD19, surface Ig +ve.
•CLL: CD5, CD19, CD23, weak surface Ig +ve.
Table 3.19 Uses of immunophenotyping
| Surface immunophenotyping | |
| DNA content of tumours | |
| TdT measurement | In leukaemias and lymphomas |
| Bone marrow transplant/stem cell transplantation | |
| Antiplatelet antibody detection | |
| Reticulocyte counts and maturation | |
| Apoptosis | |
| Detection of small numbers of cells | For example, fetal cells in mother’s circulation, microorganisms in blood |
Data from Provan Det al. Oxford Handbook of Clinical Haematology, 2nd edn, Oxford: Oxford University Press, 2004.
Particularly useful in determining whether there is a monoclonal B-cell or plasma cell population.
► Monoclonal B cells from, e.g. non-Hodgkin’s lymphoma (NHL) will have surface expression of κ or λ light chains, but not both.
► Polyclonal B cells from, e.g. a patient with infectious mononucleosis will have both κandλ expression.
Clinical Flow Wiki. http://wiki.clinicalflow.com/.
Johansson U, Bloxham D, Couzens S, et al.; British Committee for Standards in Haematology. Guidelines on the use of multicolour flow cytometry in the diagnosis of haematological neoplasms. Br J Haematol 2014; 165: 455–88.
University of Medicine and Dentistry of New Jersey. Immunophenotyping lymphomas. http://pleiad.umdnj.edu/hemepath/immuno/immuno.html.
•Looks at the number of chromosomes in each cell.
•Detects structural abnormalities between chromosome pairs.
Chromosome abnormalities may be constitutional (inherited) or acquired later in life. Cytogenetic analysis of chromosome structure and number has been used for many years for the study of disorders such as Down’s syndrome. Acquired chromosomal abnormalities are found in malignancies, especially haematological tumours. The analysis and detection of cytogenetic abnormalities is known as karyotyping. Because of the complexity of this subject area, we will concentrate on two main areas where chromosome analysis is of value.
•Prenatal diagnosis of inherited disorders:
•Detection of common aneuploidies (gain or loss of chromosomes).
•Detection/exclusion of known familial chromosome abnormalities.
•Detecting acquired chromosome abnormalities for:
•Diagnosis of leukaemia subtypes, e.g. t(15;17) characteristic of AML M3 subtype.
•Markers of prognostic information in a variety of diseases such as leukaemias, e.g. t(9;22) in acute leukaemias, N-myc amplification in neuroblastoma.
•Monitoring response to treatment (in CML, the Philadelphia chromosome t(9;22) should disappear if the malignant cells are killed).
•Haematological malignancies at diagnosis (assuming the BM is infiltrated).
•Infiltrated solid tumour tissue at diagnosis.
•Patients with equivocal morphology (e.g. type of leukaemia not clear using microscopy and other markers).
•FISH analysis when required in certain treatment protocols, e.g. MRC.
•Confirmation of disease relapse.
•Accelerated phase or blast crisis in CML.
Cytogenetic assays are expensive (around £250 for a leukaemia or lymphoma karyotype), and if there is any doubt as to whether the test is indicated, we would suggest you discuss the case with one of your seniors or the cytogenetics staff. Arranging karyotyping before or during pregnancy is generally carried out by the obstetrician in charge of the woman’s care.
(See Table 3.20.)
Table 3.20 Cytogenetic terminology
| Constitutional | Present at conception or arising during embryonic life |
| Acquired | Arise later in fetal life or after birth |
| Translocation | Exchange of material between chromosomes |
| Deletion | Loss of part of a chromosome |
| Duplication | Part of a chromosome is gained |
| Inversion | Part of a chromosome is rotated through 180° |
| Diploid | 46 chromosomes (somatic cell) |
| Haploid | 23 chromosomes (germinal cell, e.g. egg or sperm) |
| Trisomy | Extra copy of a chromosome |
| Monosomy | Loss of a chromosome |
| Aneuploidy | Loss or gain of certain chromosomes, e.g. monosomy or trisomy |
This allows both the detection of genetic diseases associated with specific chromosomal abnormalities, thereby offering the possible prevention of an affected child. With the advent of CVS in the first trimester, karyotyping can be done at an early stage of development (see Figs 3.18 and 3.19). Pre-implantation genetic diagnosis allows abnormalities to be detected even before implantation has occurred.
•Sample: amniotic fluid (15–16 weeks’ gestation).
•Biochemical tests, e.g. acetylcholinesterase.
•Sample: CVS (9–12 weeks’ gestation).
1.Cells are obtained using amniocentesis, CVS, or fetal blood sampling.
2.Cells are cultured in medium.
3.Cell division is arrested at metaphase using, e.g. colchicine.
4.Chromosomes are spread onto slides and stained.
5.Chromosomes are examined directly using light microscopy or with the aid of a computerized image analysis system.
Fig. 3.19 Normal karyotype showing metaphase chromosomes (22 autosomes 1–22, and two sex chromosomes XX or XY, depending on the sex of the patient).
Note: the banding pattern helps identify individual chromosomes, along with the position of the centromeres (the mitotic spindle attaches to these during cell division), the short (p) and long (q) arms, and telomeres (chromosome ends). (See Fig. 3.20.)
East of Scotland Regional Genetics Service.Prenatal cytogenetics. https://humangenetics.org.uk/prenatal-cytogenetics/.
Rooney DE.Human Cytogenetics, Vol 2, 2nd edn. Oxford: Oxford University Press, 2001.
University of Washington, Department of Pathology. Cytogenetics gallery. http://www.pathology.washington.edu/galleries/Cytogallery/.
•Aids the diagnosis and classification of haematological malignancy.
•Identification of prognostic groups.
•Monitoring response to therapy.
•Determining engraftment and chimerism post-allogeneic transplant.
•A normal somatic cell has 46 chromosomes: 22 pairs, and XX or XY.
•Numbered 1–22 in decreasing size order.
•Two arms meet at the centromere—short arm denoted p, long arm is q.
•Usually only visible during condensation at metaphase.
•Stimulants and cell culture used—colchicine disrupts the spindle apparatus, thereby arresting cells in metaphase.
•Chromosomes are G-banded using Giemsa or Leishman’s stain to create characteristic banding patterns along the chromosome. The regions and bands are numbered, e.g. p1, q3, etc.
•Whole chromosome gain: e.g. trisomy 8 (+8).
•Whole chromosome loss: e.g. monosomy 7 (−7).
•Partial gain: e.g. add9q+, or partial loss, e.g. del5q−.
•Translocation: material exchanged with another chromosome; usually reciprocal, e.g. t(9;22)—the Philadelphia translocation.
•Inversion: part of chromosome runs in opposite direction, e.g. inv(16) in M4Eo.
•Many translocations involve breakpoints around known oncogenes, e.g. bcr, ras, myc, bcl-2.
(See Table 3.21.)
•Molecular revolution is further refining the specific abnormalities in the genesis of haematological malignancies.
•Techniques such as FISH and PCR can detect cryptic abnormalities.
•Bcr–abl probes are now used in diagnosis and monitoring of treatment response in CML.
•IgH and T-cell receptor (TCR) genes are useful in determining clonality of suspected B- and T-cell tumours, respectively.
•Specific probes may be used in diagnosis and monitoring of subtypes of acute leukaemia, e.g. AML, e.g. PML–RARA in AML M3, t(9;22), t(12;21), and 11q23 rearrangements in paediatric ALLs.
Table 3.21 Karyotypic abnormalities in leukaemia and lymphoma
| CML | |
| t(9;22) | Philadelphia chromosome translocation creates bcr–abl chimeric gene. |
| AML | |
| t(8;21) | AML M2, involves AML–ETO gene—has better prognosis |
| t(15;17) | AML M3 involves PML–RARA gene—has better prognosis |
| inv(16) | AML M4Eo––has better prognosis |
| −5, −7 | Complex abnormalities have poor prognosis |
| MDS | |
| −7, +8, +11 | Poor prognosis |
| 5q− syndrome | Associated with refractory anaemia and better prognosis |
| Myeloproliferative disease | |
| 20q− and +8 | Common associations |
| ALL | |
| t(9;22) | Philadelphia translocation, poor prognosis |
| t(4;11) | Poor prognosis |
| Hyperdiploidy | ↑ in total chromosome number—good prognosis |
| Hypodiploidy | ↓ in total chromosome number—bad prognosis |
| T-ALL | |
| t(1;14) | Involves tal-1 oncogene |
| B-ALL and Burkitt’s lymphoma | |
| t(8;14) | Involves myc and IgH genes, poor prognosis |
| CLL | |
| +12, t(11;14) | |
| NHL | |
| t(14;18) | Follicular lymphoma, involves bcl-2 oncogene |
| t(11;14) | Mantle cell lymphocytic lymphoma, involves bcl-1 oncogene |
| t(8;14) | Burkitt’s lymphoma, involves myc and IgH genes |
Heim S, Mitelman F.Cancer Cytogenetics, 2nd edn. New York: Wisley-Liss, 1995.
Kingston HM.ABC of Clinical Genetics, 2nd edn. London: BMJ Books, 1994.
Sandberg AA.The Chromosomes in Human Cancer and Leukemia, 2nd edn. New York: Elsevier Science, 1990.
The HLA system or major histocompatibility complex (MHC) is the name given to the highly polymorphic gene cluster region on chromosome 6, which codes for cell surface proteins involved in immune recognition.
Tissue typing patients (to ensure compatibility between donor and recipient) who are undergoing transplantation to reduce the likelihood of rejection or graft-versus-host disease (GvHD) in the following types of transplant:
•These proteins are found on most nucleated cells and interact with CD8+ T lymphocytes.
•Comprises DR, DP, and DQ loci present only on B lymphocytes, monocytes, macrophages, and activated T lymphocytes.
•Class 1 and 2 genes are closely linked, so one set of gene loci is usually inherited from each parent, although there is a small amount of cross-over.
•There is ~25% chance of two siblings being HLA identical.
•There are other histocompatibility loci apart from the HLA system, but these appear less important generally, except during HLA-matched stem cell transplantation when even differences in these minor systems may cause GvHD.
Class 1 and 2 antigens were originally defined by serological reactivity with maternal antisera containing pregnancy-induced HLA antibodies. There are many problems with the technique and it is too insensitive to detect many polymorphisms. Molecular techniques are increasingly employed, such as single strand polymorphism (SSP). Molecular characterization is detecting enormous class 2 polymorphisms.
•Matching donor/recipient pairs for renal, cardiac, and marrow stem cell transplantation.
•Degree of matching more critical for stem cell than solid organ transplants.
•Sibling HLA-matched stem cell transplantation is now the treatment of choice for many malignancies.
•Unrelated donor stem cell transplants are increasingly performed, but outcome is poorer due to HLA disparity. As molecular matching advances, improved accuracy will enable closer matches to be found and results should improve.
•Mixed lymphocyte culture (MLC): now rarely used.
•Cytotoxic T-lymphocyte precursor (CTLp) assays: determine the frequency of cytotoxic T lymphocytes in the donor directed against the recipient. Provides an assessment of GvHD occurring.
•HLA on WBC and platelets may cause immunization in recipients of blood and platelet transfusions.
•May cause refractoriness and/or febrile reactions to platelet transfusions.
•Leucodepletion of products by filtration prevents this (the National Blood Service removes the WBCs at source routinely nowadays).
•Diagnosis of refractoriness confirmed by detection of HLA or platelet-specific antibodies in the patient’s serum.
•Platelet transfusions matched to recipient HLA type may improve increments.
BSBMT. Interpreting HLA typing and matching. http://bsbmt.org/registry-help-documents/.
Klein J, Sato A. The HLA system. N Engl J Med 2000; 343: 702–9. http://content.nejm.org/cgi/content/full/343/10/702?ijkey=oOilwRsUqacag.
Naik S. The human HLA system. J Indian Rheumatol Assoc 2003; 11: 79–83. http://medind.nic.in/jaa/t03/i3/jaat03i3p79.pdf.
This technique has been around since the mid 1970s. It explained much about the physical structure of genes and was a major advance in the diagnosis of many single gene disorders. The method is simple and elegant, but time-consuming. Not used as much today with the advent of PCR technology. Southern blotting relies on the physical nature of DNA whereby single strands are able to recognize and bind to their complementary sequences (see Fig. 3.21).
•Sample: EDTA sample (heparin can be used, but beware inhibitory effect on PCR amplification; if any chance PCR required, send EDTA).
1.Genomic (i.e. total) DNA is extracted from WBC in EDTA blood sample.
2.DNA is digested with bacterial restriction endonucleases (enzymes cleave DNA at specific sequences—each enzyme recognizes a different DNA sequence).
3.After digestion of the DNA, the fragments are separated on the basis of size using agarose gel electrophoresis (the smallest fragments travel the farthest).
4.The fragments are transferred to a nylon membrane and fixed permanently to the membrane using ultraviolet (UV) light.
5.Membranes are ‘probed’ using specific (known) gene probes that are radioactively labelled using 32P.
6.The location of specific binding is detected by placing the membrane next to a radiographic film (standard X-ray film).
7.The film is developed using standard techniques, and the autoradiograph generated will show bands corresponding to the position of binding of the labelled probe.
8.Fragment sizes are calculated, and the presence or absence of mutations is worked out by determining whether enzyme cutting sites have been lost through mutation.
•Historically, many diseases caused by single base changes (loss of restriction enzyme cutting site) have been diagnosed using Southern blotting.
•Sickle-cell anaemia (mutation in β-globin gene).
•Thalassaemia (mutations or deletions in α- or β-globin genes).
•Analysis of Ig or TCR genes to detect clones of cells in suspected leukaemia or lymphoma.
•Detection of chromosomal translocations in leukaemia and lymphoma (e.g. t(9;22) in CML, t(14;18) in follicular lymphoma).
Kimball’s Biology Pages. Gel blotting. http://www.biology-pages.info/G/GelBlotting.html.
Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: 503–17 (►► Southern’s classic paper and probably the most cited molecular biology paper ever).
Thermo Fisher Scientific. Southern blotting. https://www.thermofisher.com/uk/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/southern-blotting.html#.
The ability to use an enzyme to amplify specific DNA sequences has revolutionized modern diagnostic pathology. Whereas Southern blotting might take up to 1 week to produce a result, PCR can do the same thing in 2–3h! PCR is now in routine use in the analysis of oncogenes, haematological malignancies, general medicine, infectious disease, and many other specialties. Because the system amplifies the starting DNA up to a million-fold, there need only be one cell as starting material; in practice, much more DNA is required, but because of the extreme sensitivity of the technique, PCR has been used in forensic medicine where there may be only a few cells available for analysis (e.g. blood or semen stain).
(See Fig. 3.22.)
•DNA quality does not matter (can be highly degraded, e.g. with age and still be amplified—DNA from Egyptian mummies has been amplified).
•Expensive, but less so than it used to be.
•DNA sequence of the gene of interest must be known in order to design the short PCR primers (oligos). With the near completion of the Human Genome Project, this is less of a problem now.
•Highly sensitive, and contamination of samples may occur (DNA fragments float through the air constantly; if these drop into the reaction tube, a false +ve result may be obtained).
(See Fig. 3.23.)
•Two short DNA primers on either side of the gene of interest bind to the fragment of interest.
•The region between the primers is filled in using a heat-stable DNA polymerase (Taq polymerase).
•After a single round of amplification has been performed, the whole process is repeated.
•This takes place 30 times (i.e. through 30 cycles of amplification), leading to a million-fold ↑ in the amount of specific sequence.
•After the 30 cycles are complete, a sample of the PCR reaction is run on agarose gel and bands are visualized.
•Information about the presence or absence of the region or mutation of interest is obtained by assessing the size and number of different PCR products obtained after 30 cycles of amplification.
Fig. 3.22 Detection of residual leukaemia using PCR. Patients 1 and 2 have undergone chemotherapy, but as can be seen (arrow), there is still some leukaemia-specific DNA sequence present, i.e. they have minimal residual disease.
•PCR is currently used to amplify Ig genes, HIV loci, TB genes, and many other targets that are of use in molecular medicine (cystic fibrosis, haemophilia, thalassaemia, sickle-cell disease, and many others).
•PCR can be used to quantitate messenger ribonucleic acid (mRNA) species in blood samples and tissue samples. Allows gene ‘activity’ to be measured.
Nobelprize.org. The PCR method—a DNA copying machine. http://nobelprize.org/chemistry/educational/pcr/.
Principles of the PCR. http://users.ugent.be/~avierstr/principles/pcr.html.
Saiki RK. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239: 487–91 (classic PCR method paper).
Like PCR and other techniques, in situ hybridization and FISH are conceptually simple techniques that rely on the ability of a DNA probe to ‘find’ its counterpart on a chromosome and bind, and if a fluorescent tag is present, it will light up the region of binding (this modification is termed fluorescence in situ hybridization, or FISH). These techniques have evolved from standard cytogenetic analysis of metaphase chromosomes in which metaphase chromosomes were prepared on glass slides to which specific labelled probes were applied.
•Sample: discuss with your local cytogenetics or haematology laboratory (they will have specialized medium for maintaining cells from blood or marrow, so that they will divide and be suitable for hybridization studies).
The location of binding of the probe is detected by visualizing the signal produced after coating microscope slides with photographic emulsion, which generates a black area around the probe which is labelled with 32P.
A further modification based on the original principles, whereby specific gene probes are hybridized to chromosomes without the need for metaphase preparations (interphase cells can be used). Instead of 32P, the probes are labelled with a fluorescent dye and hybridization may be detected as red, blue, or other coloured dots over the cells (see Fig. 3.24).
Fig. 3.24 FISH analysis. Metaphase chromosomes are placed on a microscope slide, and the probe (e.g. for the gene of interest) is applied. The chromosome region to which the probe binds will fluoresce—highlighting its exact location in the genome.
•Used in the analysis of trisomies (chromosome gains) and monosomies (chromosome losses) associated with leukaemias and lymphomas. The presence of trisomy is detected as three fluorescent dots within the cell, whilst monosomy is seen as a single fluorescent dot within the cell.
•FISH has been used widely within paediatric leukaemias, such as ALL, where abnormalities of chromosome number are common.
Mathew P, Sanger WG, Weisenburger DD, et al. Detection of the t(2;5)(p23;q35) and NPM-ALK fusion in non-Hodgkin’s lymphoma by two-color fluorescence in situ hybridization. Blood 1997; 89, 1678–85.
O’Connor C. Fluorescence in situ hybridization (FISH). Nature Education 2008; 1: 171. https://www.nature.com/scitable/topicpage/Fluorescence-In-Situ-Hybridization-FISH-327.
Sinclair PB, Green AR, Grace C, Nacheva EP. Improved sensitivity of BCR-ABL detection: a triple-probe three-color fluorescence in situ hybridization system. Blood 1997; 90: 1395–402.
Vaandrager JW. Direct visualization of dispersed 11q13 chromosomal translocations in mantle cell lymphoma by multicolor DNA fiber fluorescence in situ hybridization. Blood 1996; 88: 1177–82.
The following laboratories provide specialized molecular, biochemical, and cellular investigations for rare haematological disorders. Please contact the laboratory before tests are requested to confirm the specimen(s) required.
National Haemoglobinopathy Reference Laboratory, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 8DU
Tel: 01865-222449; Fax: 01865-222500
E-mail: jold@hammer.imm.ox.ac.uk
Haematological Medicine, King’s College Hospital, Denmark Hill, London SES 9RS
Tel: 020-7346-1682; Fax: 020-7346-6168
E-mail: sl.thein@kcl.ac.uk
Perinatal Centre, University College Hospital, 84–86 Chenies Mews, London WC1E 6HX
Tel: 020-7388-9246; Fax: 020-7380-9864
E-mail: m.petrou@ucl.ac.uk
National Haemoglobinopathy Reference Laboratory, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 8DU
Tel: 01865-222449; Fax: 01865-222500
E-mail: jold@hammer.imm.ox.ac.uk
Department of Haematology, Central Middlesex Hospital, Acton Lane, London NW10 7NS
Tel: 020-8453-2112; Fax: 020-8965-1115
E-mail: sally.davies@dol.gso.gov.uk
Department of Haematology, Central Middlesex Hospital, Acton Lane, London NW10 7NS
Tel: 020-8453-2323
Haematological Medicine, King’s College Hospital, Denmark Hill, London SE5 9RS
Tel: 020-7737-4000 Ext 2283; Fax: 020-7346-3514
E-mail: barbara.wild@kcI.ac.uk
Haematology, ICSTM, Hammersmith Hospital, London W12 0HS
Tel: 020-8383-2173; Fax: 020-8742-9335
E-mail: m.layton@ic.ac.uk
Haematological Medicine, King’s College Hospital, Denmark Hill, London SE5 9RS
Tel: 020-7737-4000 Ext 2283; Fax: 020-7346-3514
E-mail: barbara.wild@kcl.ac.uk
Clinical Biochemistry, King’s College Hospital, Denmark Hill, London SE5 9RS
Tel: 020-7346-3856; Fax: 020-737-7434
Porphyria Service, Medical Biochemistry, University Hospital of Wales, Cardiff CF14 4XW
Tel: 02920-748349; Fax: 02920-748383
E-mail: badminton.mn@cardiff.ac.uk
Porphyria Service, Medical Biochemistry, University Hospital of Wales, Cardiff CF14 4XW
Tel: 02920-743565
International Blood Group Reference Laboratory, Southmead Road, Bristol BS10 5ND
Tel: 0117-991-2111; Fax: 0117-959-1660
E-mail: may-jean.king@nbs.nhs.uk
1 Bowden DK, Vickers MA, Higgs DR. A PCR-based strategy to detect the common severe determinants of alpha thalassaemia. Br J Haematol 1992; 81: 104–8.
2 Lewis SM, Bain BJ, Bates I (eds.). Dacie & Lewis Practical Haematology, 9th edn. Edinburgh: Churchill Livingstone, 2000.
3 Huisman TH, Carver MF. The beta- and delta-thalassemia repository. Hemoglobin 1988; 22: 169–95.
4 Serious Hazards of Transfusion (SHOT) Steering Group (2016). The 2015 annual SHOT report. https://www.shotuk.org/wp-content/uploads/SHOT-2015-Annual-Report-Web-Edition-Final-bookmarked.pdf; Annual SHOT report 2015 summary. https://www.shotuk.org/wp-content/uploads/SHOT-Summary-Infographic_Final.pdf.
