cold agglutinin – Lablogatory

Hematology Case Study: Brrrr, It’s Cold Outside!

A CBC was received on a 70-year-old surgical inpatient at our facility and was run in automated mode on our Sysmex XN analyzer. On the first run, the analyzer gave flags for RBC agglutination and MCHC >37.5. These flags require evaluation of the high MCHC with investigation of a cold agglutinin, lipemia or icterus. A smear review for RBC agglutination was also indicated. The sample was incubated at 37°C for 30 minutes and the CBC was repeated. Results are shown below in Table 1.

Table 1. CBC results before warming at 37°C and after 15 minutes incubation.

On Run 2, the MCHC is now below 37.5, so there was no operator alert to incubate further or to investigate the high MCHC. This result was validated with the comment “37°C results, possible cold agglutinin.” However, there was still an RBC agglutination flag, a high MCV, and the hemoglobin and hematocrit still don’t look great, i.e. they don’t follow ‘the rules of 3’. We know that these rules are only valid for normal samples, but if we look at the previous results from 2 days ago, the Hgb 9.3 and Hct 28.4 did follow the ‘rules of 3’ and additionally, today’s results look like the patient’s hemoglobin has not changed but the hematocrit has dropped. The RBC did go up on the second run, but it is considerably lower than 2 days ago. The indicies are also inconsistent with the previous sample. “Hmmm…What could cause this?” Instead of validating, this is what you should be asking yourself.

Cold agglutinins are known for causing pre-analytical and analytical spurious results with CBCs. With cold agglutinins, IgM antibodies bind to the RBCs after exposure to cold, causing RBC agglutination which leads to a classic pattern of false results. There is an increased MCV because the RBCs are clumped and sticking to one another, making the analyzer think these larger clumps are individual RBCs. This, in turn, will make the RBC count appear deceased because large clumps of RBCs going through the RBC aperture are counted as one RBC. The hematocrit is lowered because the volume of a clump is less than those cells individually. Hemoglobin concentration, on the other hand, is not affected by cold agglutinins, but because the hematocrit and RBC are falsely lowered, this makes the MCH and MCHC spuriously markedly increased. Cold agglutinins may be subtle, like this one, but some have extremely high MCHC and MCV, and extremely low RBC and Hct. Clues that you have a cold agglutinin are not only the high MCHC, but also flags from your analyzer such as “RBC agglutination”. The hematocrit will likely seem too low for the hemoglobin, and you may even see a hemoglobin that is higher than the hematocrit (yes it happens!) and RBC values so low as to be incompatible with life.

The first thing is to do if you get these spurious results is to compare the parameters and instrument flags and decide if the results are consistent with a cold agglutinin. Other factors that may cause a high MCHC include lipemia, icterus, low sodium, abnormal proteins, hemolyzed samples and samples from patients with hemoglobinopathies. The patterns of result with these samples will show a high MCHC, but with a normal or low MCV, and you won’t usually see an RBC agglutination flag. A sample with cold agglutinins may also appear grainy or clumpy to the naked eye. With a suspected cold agglutinin, warming the sample for 15-30 minutes will allow the RBCs to disperse and improve the results. However, these results must be reviewed, and if there are still instrument flags and/or if there is still clumping, the results may not yet be corrected or ‘correct’. Be sure to review a smear from the tube before warming and after. The first smear confirms the presence of the cold agglutinins and clumping, and the second smear should confirm the resolution of the clumping after warming.

This sample did have the characteristic grainy appearance of a cold agglutinin, and the post warming results did look a bit better, but the RBC agglutination flag was still present, and a review of the smear showed that the sample still had RBC clumping. After warming for another 30 min, the sample was quickly mixed and placed back on the analyzer. The results of this 3^rd run are shown below, in Table 2.

Figure 1. EDTA tube with RBC agglutination

Table 2. CBC results on 3 runs. Run 3, after 60 min of incubation.

Notice that hemoglobin has not changed as it is not affected by the cold agglutinins, but after warming for 60 minutes, the RBC, hematocrit and indicies all now look consistent with the previous sample drawn 2 days ago, and a review of the smear showed no RBC agglutination. These results from the 3^rd run are ready to validate.

Cold agglutinins are IgM autoantibodies that react best at 4°C but may also react at room temperature. They are generally not clinically significant and may be found in many healthy individuals. These natural cold autoantibodies occur at low titers, less than 1:64, and have no activity at higher temperatures. However, because they react at room temperature, they are notorious as a pre-analytical and analytical factor that causes spurious CBC results. They can also cause difficulties in Blood Banking during ABO/Rh typing and antibody detection.

Cold agglutinins have various clinical manifestations. Benign cold agglutinins generally do not cause hemolytic anemia and need no treatment. Most benign cold autoantibodies have anti-I specificity, are polyclonal, low titer, and do not react above 30°C. Cold agglutinins associated with Mycoplasma pneumoniae, and infectious mononucleosis are usually clinically insignificant. In cases where they do cause hemolytic anemia, the antibodies are polyclonal IgM with normal κ and λ light chains. The anemia is acute and generally spontaneously resolves in several weeks without treatment.

Though most cold agglutinins are benign and do not cause RBC destruction, when they do, they can cause hemolytic anemia that varies in severity from mild to life-threatening. This chronic cold agglutinin disease (CAD) is now known to be a form of autoimmune hemolytic anemia caused by a bone marrow lymphoproliferative disorder. Chronic CAD is a cold-autoantibody autoimmune hemolytic anemia (cAIHA) that is caused by an autoantibody produced by the clonal B cell lymphocytes. This antibody is usually monoclonal IgM with κ light chains and “I” or “i” specificity. These pathological cold agglutinins are high titer and usually react at 28°C to 32°C, and even up to 37°C. The highest temperature at which the antibodies continue to be activated is called the thermal amplitude. Because these can act at higher thermal amplitude, they may lead to CAD. In CAD the IgM autoantibodies bind to red cell antigens at 30-32°C, typically in the cooler extremities. IgM’s structure, a large immunoglobin pentamer, makes it an effective activator of the classical complement system. As the blood circulates to the central parts of the body, the RBCs warm up and the IgM antibodies dissociate from the RBC membranes, but the complement activation will continue, leading to RBC hemolysis and a cAIHA.

Chronic CAD occurs most often in adults over 50, is more common in women, and produces anemia with varying severity. Patients may be seasonally affected. In the winter, the temperature of blood may fall below 30°C in the extremities, activating the cold agglutinins. Patients may experience acrocyanosis of the hands, feet, ears, and nose with exposure to cold. They may also experience other cold related symptoms such as numbness and Raynaud’s. Patients with chronic CAD and mild anemia are therefore monitored with a ‘wait and see’ plan and advised to avoid cold temperatures. In patients with more severe anemia, it is found that targeting the underlying lymphoproliferative disorder provides the best treatment. Rituximab has been used to achieve partial remission. Therapeutic plasma exchange is also used in severe cases to rapidly remove cold agglutinins.

I have been thinking about cold agglutinins recently because of the number I have seen come into our lab this winter. As I am writing this, watching the temperature outside drop in anticipation of more snow coming in tonight, cold agglutinins came to my mind again. I used to live in the cold northern Northeast, but after moving further south, we see fewer cold agglutinins in hematology than I used to see. This winter we have had some cold spells, and, interestingly, I’ve seen more cold agglutinins. That led me to ask myself if cold agglutinin disease is really more common when patients are exposed to cold temperatures. I remember learning myself, and telling my students that that the treatment for mild to moderate CAD was to advise patients to move to someplace warmer. It has been assumed for many years that CAD worsens in colder climates or seasons. Interestingly, there have been a number of studies done since the 1950’s that examined the relationship of cold temperatures and CAD. The studies used hemoglobin, bilirubin and LDH for monitoring. Early case reports had findings that supported the theory of more anemia and higher LDH in the winter. (Dacie, Lyckholm)One recent article in 2022 found a 4-fold difference in the incidence of CAD between cold (Norway) and warm (Italy) climates. (Berentsen). However, atthe same time another study found that there was no statistically significant seasonal variation in hemoglobin, but that LDH levels were higher in winter. It concluded that these conditions should be monitored through all seasons because of the risk of hemolysis and thrombotic episodes. It was also demonstrated that though there may not be obvious statistical difference in CAD between cold and warm months, that there is a large variability of disease severity across patients and even with an individual patient. (Roth).

In conclusion, when working in any department of the laboratory, quality results are important. Results on the patient chart that vary considerably from day to day because sometimes a cold agglutinin has been effectively resolved in lab testing and other days results after 15-30 minutes of warming are just reported without a good review of the smear and the parameters, are confusing, and could affect patient care. If one tech reports the results after 30 min incubation with values that are still spurious, and the next tech resolves the agglutination with further warming, the lab will be reporting out inconsistent results. A patient who actually has stable CBC results may have deltas and what appear to be erratic results. Cold agglutinins do take time to resolve, but with over 80% of samples autoverifying with the use of auto verification, we have time to work on these problem samples. If something doesn’t look or feel right about a sample, look at all the parameters, check the instrument flags and operator alerts, check the previous results and investigate any changes. It is important to review results carefully, because we want to report out the best results possible.

References

S Berentsen, W Barcellini, S D’Sa, U Randen, THA Tvedt, B Fattizzo, E Haukås, M Kell…

Blood, The Journal of the American Society of Hematology, 2020•ashpublications.org

Climent F, Cid J, Sureda A. Cold Agglutinin Disease: A Distinct Clonal B-Cell Lymphoproliferative Disorder of the Bone Marrow. Hemato. 2022; 3(1):163-173. https://doi.org/10.3390/hemato3010014

Nikousefat Z, Javdani M, Hashemnia M, Haratyan A, Jalili A. Cold Agglutinin Disease; A Laboratory Challenge. Iran Red Crescent Med J. 2015 Oct 17;17(10):e18954. doi: 10.5812/ircmj.18954. PMID: 26566452; PMCID: PMC4636857.

Patriquin, C.J. and Pavenski, K. (2022), O, wind, if winter comes … will symptoms be far behind?. Transfusion, 62: 2-10. https://doi.org/10.1111/trf.16765

Rodak, Bernadette F., et al. Hematology: Clinical Principles and Applications. 5th ed. St. Louis, Mo., Elsevier Saunders, 2016

Röth A, Fryzek J, Jiang X, Reichert H, Patel P, Su J, et al. Complement-mediated hemolysis persists year round in patients with cold agglutinin disease. Transfusion. 2022; 62: 51–59. https://doi.org/10.1111/trf.16745

-Becky Socha, MS, MLS(ASCP)^CMBB^CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 40 years and has taught as an adjunct faculty member at Merrimack College, UMass Lowell and Stevenson University for over 20 years. She has worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. She currently works at Mercy Medical Center in Baltimore, Md. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Follow the Indicies. It’s Not Always Cold!

One of my favorite things about working in Hematology is handling those “difficult” samples. You know the ones. The one that some techs put aside to work on “later,” or they might decide it’s time to take a break when they see them coming. I love investigating and working on these interesting but perhaps uncooperative samples. At times this involves running samples in different modes, making new slides or albumin smears, and diluting samples. At other times, we investigate a delta or unusual results by checking patient diagnosis and previous results or by calling the care provider for more information and clues to help us resolve the problem.

I’m sure you’ve all seen the sayings “Without the Lab, you’re only guessing” and “Laboratory Professionals get results.” Physicians rely on the lab every day for information used to help diagnose and treat patients. Therefore, our goal is to deliver to the care provider the best possible results in a timely manner. Which means that we don’t just report results because that’s the answer the instrument gave us. With today’s instruments and middleware, we get very accurate and precise results, and about 85% or more of hematology specimens autovalidate. This is important because it leaves us time to work on those specimens with flags, and discrepancies; the ones that need a little more time and attention.

When faced with unusual or conflicting results, we first need to ask ourselves if we are dealing with a spurious sample, interfering substances or true abnormal results. Many labs today use middleware that will give the operator alerts when a sample needs to be investigated. These alerts give us suggestions as to how to handle the specimen but are usually short phrases triggered by certain values or flags and cannot be all encompassing. Operator alerts cannot tell us all the steps we may need to follow to resolve, for example, deltas, platelet clumps, abnormal scattergrams or a possible cold agglutinin. The alerts are great guidelines but it is often necessary to do more. We may need to refer to procedure manuals for SOPS or check instrument manuals or technical bulletins to decide how to handle these specimens. Sometimes we need to be detectives to report the most accurate results. We must review results with a critical eye, use all that “stuff” we learned in school, and be able to make educated decisions based on this investigation.

In my experience, one of the most common troublesome and perhaps misunderstood specimens I see is the one with a “hemoglobin (Hgb) interference” flag. An instrument flag “suspect, turbidity /Hgb interference?” is generally initiated when the MCHC is above a certain value. In our hematology lab, we see this flag when the MCHC is above 37.5 g/dL. What this is telling us is that turbidity may be present in the diluted and lysed sample. This turbidity can interfere with the Hgb detection light path and falsely increase the Hgb. Because the MCH and MCHC are calculated using the Hgb, these parameters are also affected. BUT, an MCHC >37.5 g/dL is not always something that can be or that needs to be corrected. With any parameter 95% of normal values will fall within 2SD of the mean. This means that 5% of normal healthy individuals have MCHC results <32 g/dL or >36 g/dL, and a few may have an MCHC over 37.5 g/dL. An MCHC >37.5 g/dL therefore can indicate a normal specimen, such as in a healthy young male with a Hgb at the high end of the reference range. High MCHCs can also be seen routinely in specimens from patients with spherocytosis or hemoglobinopathies such as Hgb SS, Hgb SC or Hgb C disease. In these conditions the RBCs are hyperdense due to altered surface volume and this leads to a high MCHC.

On our instrument, an MCHC >37.5 g/dL will cause a Hgb/Turbidity flag. An asterisk (*) will appear next to the Hgb, MCH and MCHC. The middleware triggers an operator alert that says “MCHC >37.5. Incubate at 37C for 30 mins. Evaluate for lipemia, icterus, hemolysis, Plasma replacement if indicated, rerun”. So, what’s the first thing to do?? Incubate? Hold on…not so fast. This is one of those instances where hematology is not just black and white. This operator alert is giving us suggestions of how to handle a specimen, but techs need to evaluate the specimen before jumping on the ‘cold’ wagon. Incubating will usually help resolve a cold agglutinin, but won’t help with a sickle cell specimen, or resolve one that’s icteric or lipemic. A grossly hemolyzed sample can give a spurious high MCHC result and, if so, needs to be recollected, not warmed. Putting a specimen that’s hemolyzed or lipemic or icteric in the heating block for 30 or more minutes would only delay reporting of results. My first case example involves a 45 year old female. The MCHC on initial run was 38.1 and the specimen gave a Hgb turbidity flag. The sample was incubated and rerun several times. After 1 hour of incubation, the MCHC was reported as 37.1 with a comment “repeated after warming for 1 hour at 37C”. In this case the patient was a known sickle cell patient. Previous results show that this patient’s MCHC is typically high and previously reported results ranged from 36.1- 37.8 g/dL. When evaluating a specimen with a high MCHC it is important to check the pattern of results. In this case the MCHC was high but the MCV was low. This does not fit the pattern for a cold agglutinin. As noted above, super dense RBCs in sickle cell patients may cause a high MCHC. This specimen was warmed, and even though the MCHC was a bit lower after warming, it would have been acceptable to report the original run MCHC. Checking patient history and previous results, and reviewing the smear for morphology would have allowed these results to be reported in a timely fashion. The operator alert does say “incubate the specimen” but it also says to evaluate. Be sure to check the MCV and MCHC along with patient history before warming specimens that don’t fit the pattern of a cold agglutinin.

Table 1. Case 1 CBC. The patient is a 45 year old known sickle cell patient.

The second example is from a 75 year old male. The CBC flagged Hgb turbidity with an MCHC of 45.8 g/dL. The MCHC >37.5 operator alert triggered Checking the pattern of results for the indicies, the MCHC was very high and the MCV was low. In a specimen with a low or normal MCV and a high MCHC, lipemia, icterus, abnormal proteins or severe leukocytosis can be affecting the Hgb. On evaluation, this sample’s Hgb and Hct did not meet the ‘rule of 3’. The rules of 3 are now generally recognized to be valid only for samples when the RBCs are normal, but the * here is telling us that there is an interference affecting the Hgb. In these cases it is valuable to know what the interference is so we know how to handle the specimen. By spinning down a small aliquot, (or asking chemistry!) we can investigate for lipemia or icterus. The specimen was found to be grossly lipemic. Flagging guidelines for lipemic specimens suggest diluting the specimen 1:5 and rerunning. Alternately, with severely lipemic or icteric samples, plasma replacement procedure may be necessary to correct the results. In this case, a plasma replacement was performed. After a plasma replacement, the WBC, RBC, Hct, MCV and platelet count are reported from the original run. The Hgb interference is what was causing the problem. Thus, when you correct the Hgb you must always correct any indicies that are calculated with the Hgb. The Hgb from the plasma replacement sample is used and the MCH and MCHC are recalculated. Notice that the new lower Hgb value now matches the Hct.

Table 2. Case 2, a 75 year old male with lipemic specimen. Plasma replacement performed. WBC, RBC, Hct, MCV, and Plt were reported from original run. Hgb was reported from plasma replacement sample. MCH and MCHC were recalculated.

Case 3 is a sample from an 80 year old woman. This was an interesting sample because there were multiple things going on here. This patient had an initial result with a high MCHC and MCH, with decreased RBC and Hct. In this patient the initial WBC was 0.64 and the RBC was 0.31. The Hgb of 9.1 /dL was less than the Hct of 3.1 %. MCV was 116 fl and the MCHC was 293.5 g/dL! In specimens with a high MCV and high MCHC we can suspect a cold agglutinin. When the MCV is very high it is because the RBCs are going through the aperture as one big bunch and this is measured as the size of one RBC. Often the Hct is less than the Hgb. Sometimes the RBC and Hct are so low that it causes the MCV to be appear within normal range. On our instrument, a RBC count of <0.5 x10⁶/μL will give a flag “abnormal RBC scattergram” but no other indicies related flags are generated, so we didn’t even get an operator alert to evaluate the MCHC. But, it’s clear there is something very wrong with these results. Warming the sample is used to loosen clumping of RBCs, which lowers the MCV and allows the RBCs to be counted. Make a smear to examine for RBC clumping and look at the sample tube. Many cold agglutinin samples will appear to be ‘grainy’ or have agglutination along the side of the tube. This is the time when we want to incubate the sample. To resolve a cold agglutinin, warming the sample is necessary. Sometime 30 minutes is enough, sometime they need to be incubated longer. Some cold agglutinins are so strong that after incubation a dilution or plasma replacement still needs to be done. Warming this sample did not lower the MCHC. After incubating, I diluted this sample, and also did a plasma replacement to see how results would compare. The new results matched. This sample took a bit more time than others but the cold agglutinin was resolved and we were able to report valid results.

Table 3. CBC results from 80 year old woman with cold agglutinin.

Image 1. Tube from cold agglutinin specimen. Note agglutination in sample along sides of tube.

There are other factors that can affect the Hct or Hgb and cause a high MCHC. Icteric specimens act much like lipemic ones and the Hgb can be corrected with dilution or a plasma replacement. An electrolyte balance can affect the Hct. Abnormal proteins and severe leukocytosis can affect the Hgb. Grossly hemolyzed samples can have a high MCHC. It is important to evaluate the indicies in these samples and correlate the values with previous results and patient history. What concerns me is that I have seen samples being warmed that do not match the indicies patterns for cold agglutinins. I have seen samples from sickle cell patients signed out with a comment “warmed at 37C. Possible cold agglutinin.” I have seen lipemic or icteric samples that are reported out with high MCHCs, erroneously high Hgb or parameters that are not reported at all. While warming these samples may actually lower the MCHC a bit, it still usually remains on the high side and does not give us the clean results that dilution or plasma replacement will. A little extra time looking at the indicies can give us important clues as to how to handle these samples. Doctors use our results every day to make patient care decisions. We need to make sure that we are making decisions every day to give them the best possible results so that patients can get the best care possible.

Table 4. Evaluating high MCHC specimens.

References

Costa, B. M. B., Vellés, M. C., Viana, M. M. F. B., & Rebelo, C. I. M. (2018). Interference of cold agglutinin autoantibodies in erythrogram interpretation: a case report and literature review. Jornal Brasileiro De Patologia e MedicinaLaboratorial, 54(4). doi: 10.5935/1676-2444.20180043
Sysmex USA. XN-Series Flagging Interpretation Guide. Document Number: 1166-LSS, Rev. 6, March 2021
It’s not Black and White: Unraveling the puzzles of Hematology. Becky Socha MS, BB, MLS(ASCP) Mercy Medical Center, Baltimore, MD