It must be understood that this query is really quite broad: e.g., the AABB Technical Methods (15 ed.) recognizes 8 different categories of positive Direct Coombs Test. Consequently, any response to the query must be related specifically to one or another of these categories, and it is impossible in this answer to cover them all. I choose to give attention primarily to three situations, namely, hemolytic disease of fetus and newborn (HDFN), incompatible transfusions, and autoimmune hemolytic anemia (AIHA).
Additionally, brief reference is here made to other Direct Coombs Test situations, which help to illuminate the subject of the query. Thus, for example, any satisfactory answer must at least allude to the following remarkable facts: Positive Direct Coombs Tests, without any evidence of hemolysis, may be encountered in (1) a very small (less than one) percent of apparently healthy blood donors; and, (2) as many as 15% of hospitalized patients (“coincidentally positive Coombs”). [Garratty, 1987] Furthermore, approximately 15 to 20% of patients taking the antihypertensive drug, alpha-methyldopa, will develop a positive Direct Coombs Test, although less than 1% of this group will demonstrate signs of hemolysis.
To assure familiarity with the terms which necessarily become involved in this discussion, I offer the following definitions:
| 1] |
Hemolysis, as an in-vivo phenomenon, refers to a shortening of the normal life span of the red cell. This fact can be established by following the disappearance of red cells labeled with radioactive isotopes, but this approach is not ordinarily taken. |
| 2] |
Hemolytic anemia, whether congenital or acquired, exists when the shortened life span of the red cell exceeds the capacity of the bone marrow to compensate for the prematurely destroyed erythrocytes. Anemia resulting from hemolysis is distinguished from other forms of anemia, such as those due to blood loss or bone marrow failure, by laboratory tests which provide evidence of (a) increased hemoglobin breakdown, (b) accompanied by bone marrow hyperactivity. Among the former tests are chemical tests on serum revealing elevation of unconjugated bilirubin, increased lactic dehydrogenase, and decreased haptoglobin. Other tests for increased hemoglobin breakdown performed on urine include those that show the presence of free hemoglobin and/or hemosiderin, elevated urobilinogen, and the absence of bilirubin (i.e., the condition recognized as “acholuric jaundice”). The single test ordinarily utilized to document augmented bone marrow activity is the reticulocyte count. Utilizing informed judgment on this array of laboratory findings, an inferential diagnosis of hemolytic anemia is usually possible. |
| 3] |
Immune Hemolytic Anemia (IHA), that form of the disease which is immunologically mediated, is usually identified when the Direct Coombs Test is positive, which can result from coating of red cells in-vivo with either immunoglobulin or complement components, or both. |
And to provide necessary background I make the following points:
| 1] |
Immune hemolysis can be caused either by alloimmune blood group antibodies, such as those responsible for hemolytic transfusion reactions and HDFN, or by autoimmune anti-red cell antibodies when they develop. |
| 2] |
An understanding of the difference between intravascular and extravascular hemolysis is critically important. |
| 3] |
The extraordinary sensitivity of the Direct Coombs Test with its ability to detect as few as 100 IgG molecules bound to a red cell surface renders it less useful for quantitating higher levels of IgG coating. |
Intravascular (extracellular) hemolysis: If a patient with a complement-fixing antibody like anti-A or some examples of anti-Jka is transfused with incompatible red cells, these cells can be destroyed extremely rapidly, right in the circulation. In these circumstances, the sudden formation of large amounts of free antigen-antibody complexes, as well as the release of red cell stroma and free hemoglobin into the circulation, may among other effects, trigger the complement cascade, leading to cardiovascular collapse (shock) and often disseminated intravascular coagulation (DIC). The rapid rise in plasma hemoglobin levels and the appearance of dark red-brown urine, as the kidneys excrete free hemoglobin, can provide the first signs of the disaster in progress. In the natural history of the problem, acute renal failure, otherwise known as kidney shutdown, is to be expected as a consequence within the next 12 to 24 hours.
An important caveat here: It might be expected in this situation that the Direct Coombs Test of the post-transfusion sample would be positive. However, depending on the volume of red cells transfused and the time interval involved, it is entirely possible that, by the time a blood sample is taken, all antibody-coated cells may be lysed, and the Direct Coombs Test may no longer be positive.
Such intravascular red cell destruction certainly fulfills the criteria for a catastrophe. This is the situation blood bankers fear the most. However, this type of hemolysis is not the subject of the present discourse.
Parenthetically, to make an important point, I will recount that I have been an anxious witness to at least 20 ABO incompatible transfusion accidents during my long blood bank career. Only one resulted in kidney shutdown. That a patient can receive large amounts of ABO incompatible blood without adverse effects if conditions are right is illustrated by a patient we transfused back in the 1970’s when I was director of the blood bank at Columbia-Presbyterian Medical Center (NYC). It involved a group O Rh Positive trauma patient admitted via the ER.
During very successful surgery, he received 24 units of group A Positive blood, as a result of an identification error in the blood bank. Apart from all-too-obvious hemoglobinuria, there were no adverse clinical effects: no shock, no kidney shutdown, no nothing. He returned to the surgical ward in stable condition. The blood bank had tried to kill him, and failed. Not to worry. His wife came into the hospital with a gun and shot him in his hospital bed. He did well again in the OR, this time correctly managed with O Positive transfusions.
Take away message: I believe kidney shutdown is very unlikely in ABO transfusion accidents if the patient is not in concomitant shock. Immediate restoration of the blood volume and blood pressure, thereby providing good perfusion of the kidneys, will greatly lessen the chance of kidney shutdown following an ABO incompatible transfusion.
Extravascular (intracellular) hemolysis: In this type of antibody-mediated hemolysis, Direct Coombs Test positive antibody-coated red cells are cleared from the circulation by the reticuloendothelial (RES) system, mainly as the coated cells pass through the spleen. Extravascular red cell destruction occurs when a non-complement fixing IgG antibody, like anti-D, coats Rh-positive cells in the circulation. The lysis of the RBC occurs intracellularly in the macrophages and monocytes, the phagocytic cells of the spleen. There is little or no extravascular lysis of RBC, and thus, little release of antigen-antibody complexes or free red cell stroma into the circulation, no rise in plasma hemoglobin, and no danger whatsoever of shock or kidney shutdown from this type of hemolysis.
Complement is not activated in these cases for two reasons: First, because at moderate concentrations of IgG Rh antibody it is unlikely that there will be sufficient antibody to enable two Rh (or other non-ABO) antibody molecules to attach to adjacent Rh (or other non-ABO) antigens on the red cell surface. In addition to this probabilistic consideration, there is the reality that Rh (or other non-ABO) antigen sites are insufficiently close together on the red cell surface to meet the requirement for geometrical-pairing of adjacent Fc fragments, as is required to activate the C1 component of complement.
As to the rate of clearance, RBCs heavily coated with Rh antibody survive in the circulation with a mean half-life of 60-100 minutes, and then 95% of such RBCs are eliminated from the circulation within 8 hours. With other antibodies Direct Coombs Test positive red cells may survive 48 hours or longer in the circulation. In some cases, Direct Coombs Test positive cells may survive normally.
The Fc piece of the antibody is all-important in the sequestration and destruction of RBC by RES macrophages and monocytes in the spleen. The Fc piece has the structural determinant of the IgG molecule responsible for complement fixation and for ensuring that particles coated or opsonized with antibody will be marked for sequestration and phagocytosis by scavenger macrophages [Gewurz, 1967]. And incidentally, a fully functioning Fc fragment on the anti-Rh IgG molecules in RhoGAM is essential for its immunosuppressive action. Therefore, the Fc fragment is critical for the specific antigen trapping and digestion of coated RBC by phagocytes of the splenic follicles. The monocyte monolayer assay (MMA), which measures by photometric or microscopic analysis the number of antibody coated erythrocytes phagocytosed by monocytes, can be a useful test for the qualitative assessment of Fc function for particular antibodies.
The essential point here is that antibody binding to red cells does not, in itself, necessarily damage the cells. Rather, a number of additional variables must be taken into account, including but not limited to, such considerations as the role of complement activation and the Fc fragment (as already alluded to), the IgG subclass and the affinity binding constant of the antibody, and whether the reticuloendothelial system’s ability to clear the red cells is saturated. All of these may affect the rate of clearance of coated cells and determine the severity of immune hemolytic disease.
The Sensitivity of the Direct Coombs Test: In general, studies show, as originally demonstrated by Mollison (1965) and confirmed by subsequent investigators, with other things being equal, a direct correlation exists between percentage antibody saturation of Rh sites and rates of in-vivo red cell clearance. At the same time, the Direct Coombs Test is too sensitive to usefully correlate with the degree of in-vivo hemolysis.
To clarify the last point, permit me to quickly summarize some quantitative aspects of the situation with some data from in vitro experiments. The Direct Coombs Test for the presence of red cell antibodies bound to the surface of washed red blood cells, e.g. anti-D bound to D antigen on Rh positive red cells, is an extremely sensitive test. For example, there are 10,000-40,000 Rh sites on a red cell, and when just 100-120 sites bind Rh antibody, amounting to less than 1% of total sites, the Direct Coombs Test becomes positive; and when over 1,000 are bound, only 3-10% of Rh sites, the Direct Coombs Test becomes 4+. Because the DC becomes strongly positive with such a low percentage saturation of sites [Stratton 1984], there is a need to use alternative quantitative tests to measure the percentage of sites bound in excess of 10%. However, tests other than the direct Coombs, such as flow cytometry or 125I-labelled anti-IgG measurements, to measure higher levels of saturation that might correlate with rate of in-vivo hemolysis, are not readily available in the clinical situation.
Thus, it is perhaps best to understand that the usefulness of the Direct Coombs Test in relation to in-vivo hemolysis as limited: the Direct Coombs Test is too sensitive to usefully correlate with the degree of hemolysis; its range is too restricted. In the evaluation of hemolysis, the Direct Coombs Test must be considered just a screening test.
I now turn to the three clinical situations where the correlation between the strength of the Direct Coombs Test and hemolysis is most at issue.
The Direct Coombs Test in Hemolytic Disease of the Fetus and Newborn:
Until relatively recently, access to Direct Coombs Tests on fetal RBC during pregnancy has been unavailable. One had to await the cord blood. Now many studies utilizing percutaneous umbilical blood sampling (PUBS) have shown the fetal cell Direct Coombs Test is strongly positive in severe cases of hemolytic disease. All this tells us is that more than 10% of Rh sites are bound. The Direct Coombs Test provides no useful correlation with the rate of hemolysis at saturation levels greater than 10%, where the Direct Coombs Test saturates.
Then, the stage of gestation, maturity of the immature fetal reticuloendothelial system, antibody transport across the placenta, the affinity binding constant of the anti-D, its IgG type and its Fc piece all may affect rate of hemolysis and determine the severity of fetal disease. Thus, the fetal Direct Coombs Test is of little interest to obstetricians performing intrauterine transfusions. Other tests such as fetal hematocrit and Doppler ultrasound of the fetal middle cerebral artery are more important in evaluating the condition of the affected fetus and guiding management.
The Direct Coombs Test in Incompatible Transfusions: Blood bankers are faced every day with patients with unexpected antibodies. It is most important to characterize the antibody. There is a spectrum of antibodies from those likely to cause immediate intravascular hemolysis to antibodies that will merely shorten the useful lifespan of transfused RBC all the way to harmless “clinically insignificant” antibodies. Here some perspective is essential in doing the best for patients. In the reports to the FDA of deaths associated with incompatible transfusions over 20 years to 2002 in the United States, there were 584 deaths reported. Most were ABO incompatibilities, and not including delayed reactions only 33 deaths over a 20-year period were associated with unexpected antibodies.
These were of anti-K, anti-Jkb, and anti-Fya specificities only, but nearly all were anti-K. Deaths associated with Rh antibodies are included, but I question these reports, as Rh antibodies do not fix complement. Perhaps high titers of IgM Rh antibody or perhaps some other clinical factor would have been found to be cause of death in these Rh cases if they were investigated closely. No other red cell antibody specificity resulted in mortality from an incompatible transfusion. [Sazama 2002].
Unexpected antibodies, uncovered in pre-transfusion serological investigations, are always a concern. However, in the calculus of probabilities involved in the decision regarding transfusion, the clear and present danger of a patient bleeding and in shock ought to greatly overweigh the slight theoretical fear of sudden intravascular hemolysis being caused by a serologically incompatible antibody. Use of red cells that can survive for several days in the presence of one of these non complement fixing antibodies may be lifesaving. The issue here is the shortened survival of transfused RBC, not kidney shutdown or transfusion related mortality.
Before leaving this subtopic, some mention must be made of the information derived from experience with delayed hemolytic transfusion reactions (DHTR). A series of reports from the Mayo Clinic have served to make it clear that most immune transfusion reactions of delayed type, revealed by the development of a positive Direct Coombs Test post-transfusion, are unaccompanied by clinical or laboratory signs of hemolysis. This experience has led to a distinction being drawn between the DHTR, in which hemolysis is discernible, and the delayed serological transfusion reaction (DSTR), in which it is not. The 19 years of Mayo Clinic experience (1980-1998) showed that 65% of the delayed reaction cases were merely DSTRs. [Pineda, 1999]
The Direct Coombs Test in Acquired Autoimmune Hemolytic Anemia (AIHA): The relevance of the Direct Coombs Test in accessing the severity of AIHA is of questionable usefulness. As already discussed, this is due partly to the relatively low levels of bound IgG necessary to give strong Direct Coombs Test agglutination when considered against the enormous amount of autoantibody seen in these cases. This combination makes the direct antiglobulin test (Direct Coombs Test) less valuable for following the progress of autoimmune hemolytic anemia than a quantitative test of how many IgG molecules are bound to the red cell surface. But, in addition, the general direct relation between the amount of bound IgG antibody and the rate of RBC destruction has many more exceptions when the antibody involved is an auto- rather than an allo- form. Perhaps even more difficult to incorporate into a reasonable understanding of AIHA is the fact that as many as 10% of the patients with warm AIHA studied by Petz and Garratty [2004] over a ten year period had negative routine Direct Coombs Tests (the phenomenon of “Coombs negative autoimmune hemolytic anemia”).
Recall that whether autoimmune anemia develops is a function of balance between the rate of red cell destruction and the ability of the bone marrow to compensate adequately through increased erythropoiesis. In AIHA there are often vast amounts of autoantibody involved. The Fc piece of the antibodies may vary. The RE system may become saturated from extensive hemolysis and limit the rate of RBC destruction. And splenectomy may be useful in tipping the balance favorably in some cases.
To summarize, any answer to the query contains some elements of a paradox, in that the correlation between Direct Coombs Test and degree of hemolysis, though theoretically present, is not necessarily straightforward.
Possibly two conclusions must simultaneously be kept in mind, namely:
| 1] |
A positive Direct Coombs Test does not always result in shortened survival of the coated RBC, and, conversely, |
| 2] |
Red cells may be prematurely destroyed via immune mechanisms when the Direct Coombs Test is negative. |
References
Garratty, G: The significance of IgG on the red cell surface. Transfusion Medicine Reviews 1987;1:47
Gewurz, H. (1967): The immunologic role of complement. Hosp. Prac., 2:45.
Skov F, Hughes-Jones NC. Observations on the number of available C antigen sites on red cells. Vox Sang. 1977 Sep;33(3):170-4.
Mollison, PL et al : Rate of removal from the circulation of red cells sensitized with different amounts of antibody. Brit J Haematology 1965;11:401.
Sazama K., Best Practices for Reducing Transfusion Errors - OBRR/CBER/FDA Workshop FDA CBER February 14, 2002 Pineda 1999
General References:
Anon: The Positive Direct Antiglobulin Test and Immune-Mediated Red Cell Destruction; Chapter 20, in Technical Manual of the AABB (15the ed); 2005; pp 453-482. Petz LD & G Garratty: Immune Hemolytic Anemias. Ed 2; Philadelphia: Churchill Livingstone, 2004.