Supplementary techniques including blood parasite diagnosis

Chapter 6 Supplementary techniques including blood parasite diagnosis





Tests for the acute-phase response


Inflammatory response to tissue injury includes alteration in serum protein concentration, especially increases in fibrinogen, haptoglobin, caeruloplasmin, immunoglobulins (Ig) and C-reactive protein (CRP), and decrease in albumin. The changes occur in acute infection, during active phases of chronic inflammation, with malignancy, in acute tissue damage (e.g. following acute myocardial infarction) with physical injury. Measurement of the acute-phase response is a helpful indicator of the presence and extent of inflammation or tissue damage and response to treatment. The usual tests are estimation of CRP and measurement of the erythrocyte sedimentation rate (ESR); some studies have suggested that plasma viscosity is also a useful indicator, but there is debate on the relative value of theses tests.1,2


Kits that are sensitive and precise are available for CRP assay; small increases in serum levels of CRP can often be detected before any clinical features become apparent, whereas as a tissue-damaging process resolves, the serum level rapidly decreases to within the normal range (<5 mg/l). The ESR is slower to respond to acute disease activity and it is insensitive to small changes in disease activity. It is less specific than CRP because it is also influenced by immunoglobulins (which are not acute-phase reactants) and by anaemia. Moreover, because the rate of change of ESR is slower than that of CRP, it rarely reflects the current disease activity and clinical state of the patient as closely as the CRP. The ESR is a useful screening test, and the conventional manual ESR method is simple, cheap and not dependent on power supply, thus making it suitable for point-of-care (near-patient) testing. It is recommended that, in clinical practice, both tests should be carried out in tandem.3 Because CRP assay is a biochemical test usually performed in the clinical chemistry laboratory, it will not be discussed further here.



Erythrocyte Sedimentation Rate


The method for measuring the ESR recommended by the International Council for Standardization in Haematology (ICSH)4 and also by various national authorities5 is based on that of Westergren, who developed the test in 1921 for studying patients with pulmonary tuberculosis. ESR is the measurement of the sedimentation of red cells in diluted blood after standing for 1 h in an open-ended glass tube of 30 cm length mounted vertically on a stand.



Conventional Westergren Method


The recommended tube is a straight glass or rigid transparent plastic tube 30 cm in length and not less than 2.55 mm in diameter. The bore must be uniform to within 5% throughout. A scale graduated in mm extends over the lower 20 cm. The tube must be clean and dry and kept free from dust. If reusable, before being reused it should be thoroughly washed in tap water, then rinsed with deionized or distilled water and allowed to dry. Specially made racks with adjustable levelling screws are available for holding the sedimentation tubes firmly in an exactly vertical position. The rack must be constructed so that there will be no leakage of the blood from the tube. It is conventional to set up sedimentation-rate tests at room temperature (18–25°C). Sedimentation is normally accelerated as the temperature increases, and if the test is to be carried out at a higher ambient temperature, a normal range should be established for that temperature. Exceptionally, when high thermal amplitude cold agglutinins are present, sedimentation becomes noticeably less rapid as the temperature is increased toward 37°C.


For the diluent, prepare a solution of 109 mmol/l trisodium citrate (32 g/l Na3C6H5O7.2H2O). Filter through a micropore filter (0.22 mm) into a sterile bottle. It can be stored for several months at 4°C but must be discarded if it becomes turbid through the growth of moulds.



Method


The method described below, originally described by the International Council for Standardization in Haematology (ICSH)4 and now adopted by the Clinical and Laboratory Standards Institute (CLSI) as its approved method,5 is intended to provide a reference method for verifying the reliability of any modification of the test.


Either collect venous blood in ethylenediaminetetra-acetic acid (EDTA) and dilute a sample accurately in the proportion of 1 volume of citrate to 4 volumes of blood, or collect the blood directly into the citrate solution. The test should then be carried out on the diluted sample within 4 h of collecting the blood, although a delay of up to 6 h is permissible provided that the blood is kept at 4°C. EDTA blood can be used within 24 h if the specimen is kept at 4°C, provided that 1 volume of 109 mmol/l (32 g/l) trisodium citrate is added to 4 volumes of blood immediately before the test is performed.


Mix the blood sample thoroughly and then draw it up into the Westergren tube to the 200 mm mark by means of a teat or a mechanical device; mouth suction should never be used. Place the tube exactly vertical and leave undisturbed for exactly 60 min, free from vibrations and draughts and not exposed to direct sunlight. Then read to the nearest 1 mm the height of the clear plasma above the upper limit of the column of sedimenting cells. The result is expressed as ESR = X mm in 1 h. A poor delineation of the upper layer of red cells may sometimes occur, especially when there is a high reticulocyte count.



Range in health


The mean values and the upper limit for 95% of normal adults are given in Table 6.1. There is a progressive increase with age, but it is difficult to define a strictly healthy population for determining normal values in individuals older than 70 years.6 In the newborn, the ESR is usually low. In childhood and adolescence, it is the same as for normal men with no differences between boys and girls. It is increased in pregnancy, especially in the later stages, and independent of anaemia;7 this is due to the physiological effect of haemodilution (an increase in the plasma volume)


Table 6.1 Erythrocyte sedimentation rate ranges in health







































Age range (years) ESR mean MM IN 1 H
10–19 8
20–29 10.8
30–39 10.4
40–49 13.6
50–59 14.2
60–69 16
70–79 16.5
80–91 15.8
Pregnancy  
Early gestation 48 (62 if anaemic)
Later gestation 70 (95 if anaemic)

In the newborn, the ESR has been reported to be 0–2 mm in 1 h, increasing to 4 mm in 1 h at 1 week, up to 17 mm in 1 h by day 14, and then 10–20 mm in 1 h for both girls and boys, until puberty.8 However, as the studies in infants were obtained by the capillary method, they are not strictly comparable to the Westergren method.



Modified methods








Sloping tube

Red cells sediment more quickly when streaming down the wall of a sloped tube. This phenomenon has been incorporated into automated systems in which the end-point is read after 20 min with the tube held at an angle of 18° from the vertical.10 Incorporating a low-speed centrifugation step (approx. 800 rpm) in this automated method reduces the end-point time further.11 These have been shown to give results comparable to the conventional method.






Semiquantitative Slide Method


Enhanced red cell adhesion/aggregation can be demonstrated by allowing a drop of citrated blood to dry on a slide. An estimate of the amount of cell aggregation on the film by image analysis provides a semiquantitative measure of the acute-phase response that appears to correlate with the ESR.14 Based on this principle, serial microscopic images of red cells aggregating on a glass slide taken every 30 s for 5 min can distinguish a normal ESR from a high value (Figs 6.1, 6.2). The images demonstrate greater spacing of cells in blood with higher ESR values compared with blood with lower ESR values.





Mechanism of Erythrocyte Sedimentation


The rate of fall of the red cells is influenced by a number of interacting factors. It depends on the difference in specific gravity between red cells and plasma, but it is influenced very greatly by the extent to which the red cells form rouleaux, which sediment more rapidly than single cells. Other factors that affect sedimentation include the ratio of red cells to plasma (i.e. the PCV), the plasma viscosity, the verticality or otherwise of the sedimentation tube, the bore of the tube and the dilution (if any) of the blood.


The all-important rouleaux formation and the red cell clumping that are associated with the increased ESR are mainly controlled by the concentrations of fibrinogen and other acute-phase proteins (e.g. haptoglobin, ceruloplasmin, α1-acid-glycoprotein, α1-antitrypsin, and CRP). Rouleaux formation is also enhanced by the immunoglobins and is retarded by albumin. Defibrinated blood normally sediments extremely slowly (i.e. not more than 1 mm in 1 h) unless the serum globulin concentration is increased or there is an unusually high globulin:albumin ratio.


Anaemia, by altering the ratio of red cells to plasma, encourages rouleaux formation and accelerates sedimentation. In anaemia, cellular factors may also affect sedimentation. Thus, in iron deficiency anaemia, a reduction in the intrinsic ability of red cells to sediment may compensate for the accelerating effect of an increased proportion of plasma.


Sedimentation can be observed to take place in three stages: a preliminary stage of at least a few minutes during which time rouleaux occur and aggregates form; then a period in which the sinking of the aggregates takes place at a constant speed; and finally, a phase during which the rate of sedimentation slows as the aggregated cells pack at the bottom of the tube. It is obvious that the longer the tube used, the longer the second period can last and the greater the sedimentation rate may appear to be.


Although ESR is a non-specific phenomenon, its measurement is clinically useful in disorders associated with an increased production of acute-phase proteins. In rheumatoid arthritis or tuberculosis it provides an index of progress of the disease, and it is of considerable value in diagnosis of temporal arteritis and polymyalgia rheumatica. It is often used if multiple myeloma is suspected, but when the myeloma is non-secretory or light chain, a normal ESR does not exclude this diagnosis.


An elevated ESR occurs as an early feature in myocardial infarction.15 Although a normal ESR cannot be taken to exclude the presence of organic disease, the vast majority of acute or chronic infections and most neoplastic and degenerative diseases are associated with changes in the plasma proteins that lead to an acceleration of sedimentation. An increased ESR in subjects who are HIV seropositive seems to be an early predictive marker of progression toward acquired immune deficiency syndrome (AIDS).16 The ESR is less helpful in countries where chronic diseases are rife; however, one study has shown that very high ESRs (higher than 100 mm/h) have a specificity of 0.99 and a positive predictive value of 0.9 for an acute or chronic infection.17 The ESR is influenced by age, stage of the menstrual cycle and medications taken (corticosteroids, contraceptive pills). It is especially low (0–1 mm) in polycythaemia, hypofibrinogenaemia and congestive cardiac failure and when there are abnormalities of the red cells such as poikilocytosis, spherocytosis, or sickle cells. In cases of performance-enhancing drug intake by athletes (discussed below) the ESR values are generally lower than the usual value for the individual and as a result of the increase in haemoglobin (i.e. the effect of secondary polycythaemia).



Plasma Viscosity


The ESR and plasma viscosity generally increase in parallel with each other.1 Plasma viscosity is, however, primarily dependent on the concentration of plasma proteins, especially fibrinogen, and it is not affected by anaemia. Changes in the ESR may lag behind changes in plasma viscosity by 24–48 h, and viscosity seems to reflect the clinical severity of disease more closely than does the ESR.18


There are several types of viscometers, including rotational and capillary types that are suitable for routine use1 and, as for ESR methods, automated closed-tube methods are available.19 The main use of plasma viscosity is in the investigation of individuals with suspected hyperviscosity, myeloma and macroglobulinaemia. In conjunction with the ESR and CRP, the plasma viscosity can be used as a marker for inflammation. The viscosity test should be carried out as described in the instruction manual for the particular instrument used.



Reference Values


Each laboratory should establish its own reference values for plasma viscosity. As a general guide, ICSH has recorded that with the Harkness capillary viscometer normal plasma has a viscosity of 1.16–1.33 mPa/s (if expressed in poise (P), 1 cP = 1 mPa/s) at 37°C and 1.50–1.72 mPa/s at 25°C.20 Plasma viscosity is lower in the newborn (0.98–1.25 mPa/s at 37°C), increasing to adult values by the 3rd year; it is slightly higher in old age. There are no significant differences in plasma viscosity between men and women or in pregnancy. It is remarkably constant in health, with little or no diurnal variation, and it is not affected by exercise. A change of only 0.03–0.05 mPa/s is thus likely to be clinically significant.




Heterophile antibodies in serum: diagnosis of infectious mononucleosis


Infectious mononucleosis (IM) is caused by Epstein–Barr virus.22 The immune response that develops in response to virus-infected cells includes not only antibodies to viral antigens but also characteristic heterophile antibodies. Before the nature of this reaction was understood, Paul and Bunnell23 demonstrated the antibodies as agglutinins directed against sheep red cells. They are, in fact, not specific for sheep red cells but also react with horse and ox, but not human, red cells. They are IgM globulins, which are immunologically related to, but distinct from, antibodies that occur in response to the Forssman antigens. The latter are widely spread in animal tissue; they occur at low titre in healthy individuals and at high titre in serum sickness and in some leukaemias and lymphomas.24,25 In these non-IM conditions, the antibody can be absorbed out by guinea pig cells. Thus, for the diagnosis of IM, it is necessary to demonstrate that the antibody present has the characters of the Paul–Bunnell antibody (i.e. it is absorbed by ox red cells but not by guinea pig kidney). This is the basis of the absorption tests for IM (‘monospot’ test). Immunofluorescent antibody tests have been developed to distinguish the IgM antibody, which occurs at high titre in the early phase of IM and diminishes during convalescence, from the IgG antibody, which persists at high titre for years after infection26,27 and which also occurs in the non-IM infections.22,28



Screening Tests for Infectious Mononucleosis


The reagents for IM screening are available commercially in diagnostic kits from several manufacturers. Guinea pig cells can be also be manufactured locally as described in previous editions. Some kits are based on agglutination of stabilized horse red cells or antigen-coated latex particles to which IM antibody binds. An extensive evaluation of 14 slide tests for the UK Medical Devices Agency (MDA), showed them to have a sensitivity between 0.87 and 1.00 and specificity of 0.97 to 1.00, with an overall accuracy (positive and negative) in the order of 91–100%.25 False-positive reactions have been reported in malaria, toxoplasmosis, and cytomegalovirus infection; autoimmune diseases; and even occasionally without any apparent underlying disease.29,30 False-negative reactions occur if the test is carried out before the level of heterophile antibody has increased or conversely when it has decreased. False-negative reactions may also occur in the very young and the very old. In the UK MDA study the best performance was obtained with the Clearview test (Unipath

Only gold members can continue reading. Log In or Register to continue

Stay updated, free articles. Join our Telegram channel

Jun 12, 2016 | Posted by in HEMATOLOGY | Comments Off on Supplementary techniques including blood parasite diagnosis

Full access? Get Clinical Tree

Get Clinical Tree app for offline access