If haemolytic anaemia is suspected, a full blood count, reticulocyte count and blood film should always be performed. The blood count shows a reduced haemoglobin concentration (Hb) and, usually, an increased mean cell volume (MCV). The increased MCV is attributable to the fact that reticulocytes, which may constitute a significant proportion of total red cells, are larger than mature red cells. The abnormalities that may be detected in the blood film and their possible significance in acquired haemolytic anaemia are shown in Table 13.1. Abnormalities detected in the blood film will direct further investigations. For example, a Heinz body preparation would be relevant if irregularly contracted cells were present, particularly if there appeared to be red cell inclusions. Similarly, a direct antiglobulin test (DAT) would be indicated if the blood film showed spherocytes. Various inherited forms of haemolytic anaemia enter into the differential diagnosis of suspected acquired haemolytic anaemia. Thus, spherocytes could be attributable to hereditary spherocytosis as well as to autoimmune or alloimmune haemolytic anaemia. Haemolysis with irregularly contracted cells could be attributable not only to oxidant exposure but also to an unstable haemoglobin, homozygosity for haemoglobin C or glucose-6-phosphate dehydrogenase (G6PD) deficiency.

Table 13.1 Abnormalities that may be detected on blood film examination and their possible significance

Morphological abnormality observed on blood film examination	Type of acquired haemolytic anaemia suggested
Schistocytes	Fragmentation syndromes including microangiopathic haemolytic anaemia and mechanical haemolytic anaemia
Spherocytes	Autoimmune, alloimmune or drug-induced immune haemolytic anaemia, paroxysmal cold haemoglobinuria, burns, Clostridium perfringens sepsis
Microspherocytes	Burns, fragmentation syndromes
Irregularly contracted cells	Oxidant damage, Zieve’s syndrome
Ghost cells, hemi-ghosts and suspicion of Heinz bodies	Acute oxidant damage
Marked red cell agglutination	Cold-antibody-induced haemolytic anaemia
Minor red cell agglutination	Warm autoimmune haemolytic anaemia, paroxysmal cold haemoglobinuria
Red cell agglutination plus erythrophagocytosis	Particularly characteristic of paroxysmal cold haemoglobinuria
Hypochromia, microcytosis and basophilic stippling	Lead poisoning
Erythrophagocytosis	Paroxysmal cold haemoglobinuria
Atypical lymphocytes	Cold-antibody-induced haemolytic anaemia associated with infectious mononucleosis or, less often, other infections
Lymphocytosis with mature small lymphocytes and smear cells	Autoimmune haemolytic anaemia associated with chronic lymphocytic leukaemia
Thrombocytopenia	Autoimmune haemolytic anaemia (Evans’ syndrome), thrombotic thrombocytopenic purpura, microangiopathic haemolytic anaemia associated with disseminated intravascular coagulation, paroxysmal nocturnal haemoglobinuria
Neutropenia	Paroxysmal nocturnal haemoglobinuria
No specific red cell features	Paroxysmal nocturnal haemoglobinuria

Immune haemolytic anaemias

Acquired immune-mediated haemolytic anaemias are the result of autoantibodies to a patient’s own red cell antigens or alloantibodies in a patient’s circulation, either present in the plasma or completely bound to red cells (e.g. transfused or neonatal red cells). Alloantibodies may be present in a patient’s plasma and react with antigens on transfused donor red cells to cause haemolysis. Alloantibodies may also occur in maternal plasma and cause haemolytic disease of the newborn. Autoimmune haemolytic anaemia (AIHA) may be ‘idiopathic’ or secondary, associated mainly with lymphoproliferative disorders and autoimmune diseases, particularly systemic lupus erythematosus. AIHA may also follow atypical (Mycoplasma pneumoniae) pneumonia or infectious mononucleosis and other viral infections. AIHA has also been reported following allogeneic bone marrow transplantation ¹ and other hematopoietic stem cell transplantation in both adult² and paediatric patients.³ Paroxysmal cold haemoglobinuria (PCH) also belongs to this group of disorders. Occasionally, drugs may give rise to a haemolytic anaemia of immunological origin that closely mimics idiopathic AIHA both clinically and serologically. This was a relatively common occurrence with α-methyldopa, a drug that is now used very infrequently, but it also occurs occasionally with other drugs. A larger range of drugs give rise to an antibody that is directed primarily against the drug and only secondarily involves the red cells. This is an uncommon occurrence. Such drugs include penicillin, phenacetin, quinidine, quinine, the sodium salt of p-aminosalicylic acid, salicylazosulphapyridine and cephalosporins.⁴

Types of Autoantibody

The diagnosis of an AIHA requires evidence of anaemia and haemolysis and demonstration of autoantibodies attached to the patient’s red cells (i.e. a positive DAT, see p. 279). A positive DAT may also be caused by the presence of alloantibodies (e.g. owing to a delayed haemolytic transfusion reaction), so details of any transfusion in the past months must be sought.

Autoantibodies can often be demonstrated free in the serum of a patient suffering from an AIHA. The ease with which the antibodies can be detected depends on how much antibody is being produced, its affinity for the corresponding antigen on the red cell surface and the effect that temperature has on the adsorption of the antibody, as well as on the technique used to detect it. The autoantibodies associated with AIHA can be separated into two broad categories depending on how their interaction with antigen is affected by temperature: warm antibodies, which are able to combine with their corresponding red cell antigen readily at 37°C and cold antibodies, which cannot combine with antigen at 37°C but form an increasingly stable combination with antigen as the temperature falls from 30–32°C to 2–4°C.

Cases of AIHA can similarly be separated into two broad categories according to the temperature characteristics of the associated autoantibodies: warm-type AIHA and the less frequent cold-type AIHA. The relative frequency of the two categories is illustrated in Table 13.2.⁵ In unusual instances, both warm autoantibody and cold autoantibody are detected in the patient’s serum and those cases are referred to as mixed-typed AIHA. This can be further classified into idiopathic or secondary, the latter often associated with systemic lupus erythematosus or lymphoma.^6,⁷

Table 13.2 Relative incidence of different types of autoimmune haemolytic anaemia ⁵

Warm Autoantibodies

The most common type of warm autoantibody is an immunoglobulin (Ig) G, which behaves in vitro very similarly to an Rh alloantibody; indeed, many IgG autoantibodies have a mimicking Rh specificity. IgA and IgM warm autoantibodies are much less common and when present they are usually formed in addition to an IgG autoantibody (Table 13.3).⁸

Table 13.3 Direct antiglobulin test in warm-antibody autoimmune haemolytic anaemia: incidence of different reactions to specific antiglobulin sera ⁸

Frequently, patients with warm-type AIHA have complement adsorbed onto their red cells and the red cells are therefore agglutinated by antisera specific for complement or a complement component such as C3d (Table 13.3). In these cases, the complement is probably not being bound by an IgG antibody but is on the cell surface as the result of the action of small and otherwise undetected amounts of IgM autoantibody.

IgG can fix complement and sometimes patients with warm-type AIHA appear to have a positive DAT with complement components only on the red cell surface. Similar results (positive DAT with complement only) are seen in some patients with no evidence of increased red cell destruction, due to binding of circulating immune complexes to the red cells.

Warm autoantibodies free in the patient’s serum are best detected by means of the indirect antiglobulin test (IAT) or by the use of enzyme-treated (e.g. trypsinized or papainized) red cells. (Antibodies that agglutinate unmodified cells directly in vitro are seldom present.) Not infrequently, antibodies that agglutinate enzyme-treated cells, sometimes at high titres, are present in the sera of patients in whom the IAT using unmodified cells is negative (Table 13.4). Occasionally, too, they are present in the sera of patients in whom the DAT is negative.

Table 13.4 Results of testing for free autoantibodies in the sera of 210 patients with warm-antibody autoimmune haemolytic anaemia ⁵

Antibodies in serum that can be shown to lyse (rather than simply agglutinate) unmodified red cells at 37°C in the presence of complement (warm haemolysins) are rarely demonstrable. If they are present, the patient is likely to suffer from extremely severe haemolysis. Antibodies in serum that lyse as well as agglutinate enzyme-treated cells but do not affect unmodified cells are, however, quite common. Their specificity is uncertain – they are not anti-Rh – and their presence is not necessarily associated with increased haemolysis.

Cold Autoantibodies

Cold autoantibodies are nearly always IgM in type. In vivo the majority do not cause haemolysis, although a minority can cause chronic intravascular haemolysis, the intensity of which is characteristically influenced by the ambient temperature. The resultant clinical picture is generally referred to as the cold haemagglutinin syndrome or disease (CHAD). Haemolysis results from destruction of the red cells by complement that is bound to the red cell surface by the antigen–antibody reaction, which takes place in the blood vessels of the exposed skin where the temperature is 28–32°C or less. The cold autoantibody in CHAD is monoclonal because this syndrome is the result of a low-grade lymphoproliferative disorder.

The red cells of patients suffering from CHAD characteristically give positive antiglobulin reactions only with anticomplement (anti-C′) sera. (The C′ notation is used to distinguish anticomplement antibodies from anti-C antibodies of the Rh system.) This is because of the presence of red cells that have irreversibly adsorbed sublytic amounts of complement; it is an indication of an antigen–antibody reaction that has taken place at a temperature below 37°C. The complement component responsible for the reaction with anti-C′ sera is the C3dg derivative of C3 (see p. 494).

In vitro, a cold-type autoantibody will often lyse normal red cells at 20–30°C in the presence of fresh human complement, especially if the cell-serum mixture is acidified to pH 6.5–7.0; it will usually lyse enzyme-treated red cells readily in unacidified serum and agglutination and lysis of these cells may still occur at 37°C. Most of these cold-type autoantibodies have anti-I specificity (i.e. they react strongly with the vast majority of adult red cells and only weakly with cord-blood red cells). A minority are anti-i and react strongly with cord-blood cells and weakly with adult red cells. Rarely, the antibodies have anti-Pr or anti-M specificity and react with antigens on the red cell surface that are destroyed by enzyme treatment.

Combined Warm and Cold Autoantibodies

In approximately 7% of cases with AIHA, both warm IgG antibody and cold IgM autoantibody are simultaneously detected in the patient’s serum.^6,⁷ These cases are referred to as ‘combined warm and cold AIHA’ or mixed-type AIHA. The serological characteristics in these patients are the presence of IgM cold autoantibody with a high thermal amplitude (reacting at or above 30°C) in association with a warm IgG autoantibody. In some cases, high- titre cold agglutinins (>1024 at 4°C) were reported^9,¹⁰ and in others the cold agglutinin titre were reported as >64 at 4°C.^11,¹²

Another quite distinct, but rarely encountered, type of cold antibody is the Donath–Landsteiner (D–L) antibody. This is IgG and has anti-P specificity. The clinical syndrome the antibody produces is PCH.

PCH is caused by a biphasic IgG autoantibody, usually with anti-P specificity, and is commonly seen as an acute condition in children. This antibody binds to the red cells in the cold but activates complement and causes haemolysis on rewarming to 37°C. Cases may be idiopathic or can be secondary to acute viral infection in children. Other tests of value in the diagnosis of PCH are discussed on p. 287.

The DAT is positive for complement only. A negative antibody screen by the standard IAT at 37°C is a common finding in a suspected case of PCH because of the low thermal amplitude of the autoantibody. If the antibody investigation is carried out at a lower temperature in PCH cases, panreactive cold antibodies may be detected because the majority of autoantibodies show anti-P specificity with thermal amplitude range up to 15–24°C. Usually the antibody titre is low (<64), even when investigated at 4°C.

Some of the characteristics of IgG, IgM and IgA antibodies are listed in Table 13.5.

Table 13.5 Main characteristics of IgG, IgM and IgA autoantibodies

The clinical, haematological and serological aspects of the AIHAs have been summarized by Dacie ¹³ and others.¹⁴^–¹⁸

Methods of Investigation

Many of the methods used in the investigation of a patient suspected of suffering from AIHA are described in Chapter 21. Detailed description is given here of precautions to be taken when collecting blood samples from patients and of methods of particular value in the investigations.

Collection of Samples of Blood and Serum

To determine the true thermal amplitude or titre of cold agglutinins requires that the blood sample is collected and maintained strictly at 37°C until serum and cells are separated. This can be achieved by collecting venous blood (clotted and ethylenediaminetetra-acetic acid [EDTA]-anticoagulated samples) and keeping it warmed at 37°C – ideally in an insulated thermos, but usually, in practice, by placing the sample tube in a beaker containing water at 37°C.

The red cells are available for antibody elution and the serum can be examined for free antibody or other abnormalities. The clotted sample should then be centrifuged to separate the serum at 37°C (e.g. in an ordinary centrifuge into the buckets of which has been placed water warmed to 37–40°C). The EDTA sample is used for the DAT and other tests involving the patient’s red cells. If the autoantibody in a particular case is known to be warm in type, the blood may be separated at room temperature; otherwise, as already indicated, this should be carried out at 37°C. When samples are sent by post, it is best to send separately: (1) serum (separated at 37°C) and (2) whole blood added to acid–citrate–dextrose (ACD) or citrate–phosphate–dextrose (CPD) solution. Sterility must be maintained.

Storage of Samples

Samples of a patient’s blood, while keeping quite well in ACD or CPD at 4°C, are more difficult to preserve than normal red cells. In particular, if marked spherocytosis is present, considerable lysis develops on storage. However, satisfactory eluates can be made from washed red cells that are frozen at –20°C for weeks or months.

The patient’s serum should be stored at –20°C or below in small (1–2 ml) volumes. If complement is to be titrated and the titration is not performed immediately, the serum should be frozen as soon as practicable at −70°C or below.

Scheme for Serological Investigation of Haemolytic Anaemia Suspected to be of Immunological Origin

It is important to consider which are the most useful tests to carry out and the order in which they should be done. A suggested scheme has been set out in the form of answers to questions.¹⁹ Whereas some information may be helpful in classifying the type of AIHA, the single most important practical consideration is to determine whether, in addition to an autoantibody, there is any underlying alloantibody present. This should be identified before transfusion is undertaken to avoid a delayed haemolytic transfusion reaction that would compound existing haemolysis.

1. Are the patient’s red cells ‘coated’ by immunoglobulins or complement (indicating an antigen–antibody reaction)?

Perform a DAT using a polyspecific ‘broad-spectrum’ reagent, which contains both anti-IgG and anti-C′. (If the DAT is negative, it is unlikely, although not impossible, that the diagnosis is AIHA. See DAT-negative AIHA, p. 281)

2. If the DAT is positive, are immunoglobulins or complement adsorbed to the red cells?

Repeat the DAT using monospecific sera (see p. 500) (i.e. anti-IgG and anti-C3d).

3. If immunoglobulins are present on the red cells, is there antibody specificity?

Prepare eluates from the patient’s red cells. Test these later (see item 6).

4. What is the patient’s blood group?

Determine the patient’s ABO and RhD and Kell type. The Rh phenotype is particularly important in warm-type AIHA; other antigens must be determined if alloantibodies are to be differentiated from autoantibodies (see p. 507).

5. Is there free antibody in the serum? How does it react and at what temperatures and by what methods can it be demonstrated? Is there any underlying alloantibody present?

Screen the serum with two or three red cell suspensions suitable for routine pretransfusion antibody screening (see p. 528) looking for agglutination and lysis at 37°C by the IAT (see p. 500). If positive, identify the antibody using an antibody identification panel.

a. If an alloantibody is identified, blood lacking the corresponding antigen must be selected for transfusion.

b. If no alloantibody is identified in the serum or plasma, it is safe to assume there is no alloantibody present, unless the patient has been transfused in the last month; in the latter case, a red cell eluate is required because an alloantibody may be bound to the recently transfused cells and there may not be free antibody detectable in the serum/plasma.

c. If the autoantibody is pan-reacting (i.e. is reacting against all panel cells), antibody adsorption tests are needed to remove the autoantibody so as to identify any underlying alloantibody. If the patient has not had a transfusion within the last 3 months, a ZZAP autoadsorption test is appropriate (see p. 283). If the patient has had a transfusion within the last 3 months, differential alloadsorption tests are needed. However, if the patient has had a transfusion within the last month, an eluate is required, irrespective of results of adsorption tests.

6. If there is a warm autoantibody, what is the specificity of the autoantibody?

Test the serum also at 20°C against antibody-screening cells to show whether cold or warm antibodies or a mixture of the two, are present in the serum.

Test the eluate against the antibody identification panel of red cells by IAT and by using enzyme-treated red cells (see p. 498). Titration of autoantibody may be useful in the presence of a strong alloantibody.

Titrate the serum/plasma by the methods that have given positive results in the screening test using the same panel of red cells (see item 5a).

7. If there is a cold antibody:

a. Has the antibody any specificity? Is it an autoantibody or an alloantibody? What is its titre?

b. What is the thermal range of the antibody?

Test the serum/plasma against a panel of O cells, O cord cells and patient’s own cells at 20°C. If an autoantibody is found, titrate at 4°C with ABO-compatible adult (I) cells, cord blood (i) cells, the patient’s cells and adult (i) cells (if possible):

i. Determine the highest temperature at which autoagglutination of the patient’s whole blood takes place (see p. 287).

ii. Titrate the patient’s serum/plasma at 20°C, 30°C and 37°C with pooled O adult cells, O cord cells, patient’s own cells and the panel of cells described earlier. If there was any agglutination or lysis at 37°C in the screening test (item 5), titrate with the appropriate cells at this temperature.

iii. If PCH is suspected, carry out the direct and two-stage indirect Donath–Landsteiner tests (see p. 287).

8. Is there is a mixture of both warm autoantibody and cold autoantibody?

The diagnosis of mixed-type AIHA can only be made after appropriate characterization of the serum autoantibodies. The serological characteristic in these cases is the presence of a cold IgM antibody with a high thermal amplitude (reacting at or above 30°C) in association with a warm IgG autoantibody.^6,⁹

9. Is a drug suspected as the cause of the haemolytic anaemia?

a. If a penicillin-induced haemolytic anaemia is suspected, test for antibodies using cells pre-incubated with penicillin (see p. 290).

b. If haemolysis induced by other drugs is suspected, add the drug in solution to a mixture of the patient’s serum, normal cells and fresh normal serum (see p. 290). Look for agglutination of normal and enzyme-treated cells and use the IAT.

10. Are there any other serological abnormalities?

Consider carrying out the following tests: serum protein electrophoresis and quantitative estimation of immunoglobulins, estimation of complement, tests for antinuclear factor, a screening test for heterophile antibodies (infectious mononucleosis screening test) and a test for mycoplasma antibodies.

Detection of Incomplete Antibodies by Means of the Direct Antiglobulin (Coombs) Test

Szymanski et al.²⁵ used an AutoAnalyser and used Ficoll and polyvinylpyrrolidone (PVP) to enhance agglutination by an anti-IgG serum highly diluted (usually to 1 in 5000) in 0.5% bovine serum albumin. In this sensitive system, the strength of agglutination was positively correlated with the serum γ-globulin concentration, being subnormal in hypogammaglobulinaemia and supranormal in hypergammaglobulinaemia.²⁶

Usually, in patients with hypergammaglobulinaemia in whom the DAT is positive, attempts to demonstrate antibodies in eluates fail (i.e. eluates are non-reactive).^27,²⁸

9. Cross-reacting antiphospholipid antibodies adsorbing non-specifically on to red cell membrane and binding to phospholipid-dependent epitopes. Positive DAT as a result of antiphospholipid antibodies has been documented in patients with primary antiphospholipid syndrome and antiphospholipid syndrome with systemic lupus erythematosus.²⁹ It has also been described in healthy blood donors.³⁰

10. Incidental findings of positive DAT with no clear correlation between DAT and anaemia. A positive DAT is a common finding in patients with sickle cell disease because of abnormal amounts of IgG coating red cells. There was no correlation between the amount of IgG on the patient’s red cells and the severity of anaemia.³¹

There was an association demonstrated between an elevated blood urea nitrogen and a positive DAT.³² Urea may alter the red cell membrane and enhance non-specific IgG adsorption.³²

11. Sensitization in vitro if a sample other than EDTA is used. If, for instance, clotted or defibrinated normal blood is allowed to stand in a refrigerator at 4°C or even at room temperature, and the antiglobulin test is subsequently carried out, the reaction may be positive because of the adsorption of incomplete cold antibodies and complement from normal sera. Samples of blood taken into EDTA or ACD and subsequently chilled do not give this type of false-positive result because the anticoagulant inhibits the complement reaction.

12. False-positive agglutination may occur with a silica gel derived from glass.³³ Also, albeit rarely, the DAT has been positive with the blood of apparently perfectly healthy individuals (e.g. blood donors). Such occurrences have not been satisfactorily explained (see below).

Positive DATs in Normal Subjects

The occurrence of a clearly positive DAT in an apparently healthy subject is a rare but well-known phenomenon. Worlledge ²⁰ reported a prevalence in blood donors of approximately 1 in 9000. In a later report, Gorst et al.³⁴ estimated that the prevalence was approximately 1 in 14 000 with an increasing likelihood of a positive test with increasing age. Their report and subsequent reports,^25,³⁵ suggest that the finding of a positive DAT, using an anti-IgG serum, in an apparently healthy person is usually of little clinical significance and that, although overt AIHA may subsequently develop, this is infrequent. In some such individuals the DAT eventually becomes negative.

Positive DATs in Hospital Patients

In contrast to the rarity of positive DATs in healthy people, positive tests are much more frequent in hospital patients. Worlledge ²⁰ reported that the red cells of 40 out of 489 blood samples (8.9%) submitted for routine tests were agglutinated by anti-C′ sera. Only one sample was agglutinated by an anti-IgG serum and this had been obtained from a patient being treated with α-methyldopa. Freedman³⁶ reported a similar incidence – 7.8% positive tests with anti-C′ sera. Lau et al.³⁷ used anti-IgG sera only. The tests were seldom positive (0.9% positive out of 4664 tests). The probable explanation for the relatively high incidence of positive tests with anti-C′ sera is that the reaction is between anti-C′ antibodies and immune complexes adsorbed to the red cells.

False-Negative Antiglobulin Test Results

There are several causes of false-negative test results:

1. Failure to wash the red cells properly: the antisera may then be neutralized by immunoglobulins or complement in the surrounding serum or plasma (see p. 501).

2. Excessive agitation at the reading stage: this may break up agglutinates, leading to a false-negative result.

3. The use of impotent antisera so that weakly sensitized cells are not detected.

4. The use of antisera lacking the antibody corresponding to the subclass of immunoglobulin responsible for the red cell sensitization.

5. The presence of an antibody that is readily dissociable and is eluted in the washing process.

These phenomena are largely negated by the use of column agglutination technology.

DAT-Negative Autoimmune Haemolytic Anaemia

Most hospital blood banks use polyspecific ‘broad-spectrum’ AHG reagents for screening for diagnosis of AIHA. These reagents contain antibody to human IgG and the C3d component of human complement and have little activity against IgA and IgM proteins. The incidence of IgA-only warm AIHA has been reported as 0.2% to 2.7%,³⁸ and the diagnosis may be missed if such polyspecific AHG is used for the DAT screen. In approximately 2–6% of patients who present with the clinical and haematological features of AIHA, the DAT is negative on repeated testing.^20,^39,⁴⁰

Low-affinity IgG autoantibodies dissociate from the red cells during the washing phase if a tube technique is used, resulting in a negative DAT. Alternatively, there may be few IgG molecules coating the red cells and this number may fall below the threshold of detection, which is 300–4000 molecules per red blood cell if a tube technique is used. In such cases, a positive DAT may be demonstrated by a more sensitive technique, such as a column agglutination method, an enzyme-linked immunoabsorbant assay or flow cytometry.⁴¹^–⁴³

If polyspecific AHG is used and the DAT remains negative with clinical evidence of haemolysis, a more sensitive technique should be used for further investigation.⁴⁴

The DiaMed DAT gel card, which contains a set of monospecific AHG reagents (i.e. anti-IgG, -IgA, -IgM, -C3c, -C3d and an inert control) can be used. Because there is no washing phase, this permits the detection of low-affinity IgG, IgA and IgM antibodies. A gel card can also pick up the rare IgA-only autoimmune haemolytic anaemia. In warm-type AIHA the DAT may be positive with anti-IgG or anti-IgG plus anti-C3d. In cold–type AIHA the DAT may be positive with anti-IgM or anti-IgM plus anti-C3d and in mixed-type AIHA the DAT may be positive with anti-IgG, anti-IgM and anti-C3d.

Preparing and Testing a Concentrated Eluate

The eluate technique concentrates low levels of immunoglobulin present on the red cell surface so that antibody may then be detected by screening the eluate with group O red cells by the IAT. Elution techniques reverse or neutralize the binding forces that exist between the red cell antigens and the antibody coating the cells. This may be achieved by several techniques (e.g. heat or alterations to the pH).

Manual Direct Polybrene Test

The following method ⁴⁵ is modified from that of Lalezari and Jiang.⁴⁶ Polybrene is a polyvalent cationic molecule, hexadimethrine bromide, that can overcome the electrostatic repulsive forces between adjacent red cells, bringing the cells closer together. When low levels of IgG are present on the red cell surface, antibody linkage of adjacent red cells is enhanced. The Polybrene is then neutralized using a negatively charged molecule such as trisodium citrate. Sensitized red cells remain agglutinated after neutralization of the Polybrene. Unsensitized red cells will disaggregate after neutralization.